Immunotherapeutic molecules and uses

ABSTRACT

The invention provides molecule comprising:
         (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and   (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell,   wherein the masked immune cell binding region is capable of being selectively unmasked when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell.

This is a divisional of application Ser. No. 14/381,405, which is anational stage entry from PCT/GB2013/050499, filed on Feb. 28, 2013, andwhich claims priority to UK application number GB1203442.7, filed onFeb. 28, 2012.

This is a continuation of application Ser. No. 14/381,405, which is anational stage entry from PCT/GB2013/050499, filed on Feb. 28, 2013, andwhich claims priority to UK application number GB1203442.7, filed onFeb. 28, 2012.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith: File name: 20151014_01131-0002-01US_SeqList; created Oct. 14,2015, 17 KB in size. The content of the sequence listing informationrecorded in the computer-readable form is identical to the writtensequence listing and includes no new matter.

The present invention relates to immunotherapeutic molecules. Inparticular, it relates to immunotherapeutic molecules that can be usedto prevent or treat a condition characterised by the presence ofunwanted cells, such as tumours or other disease causing cells.

Immunotherapeutic strategies for targeting malignant disease are anactive area of translational clinical research, and have been forseveral decades. The current models dictate that cancer representseither a functional or constitutional immunodeficiency which can betreated with immunotherapeutic manipulation of the host. These effortscan be broadly classified into two groups. The first serves to augmentor support endogenous anti-tumour immunity through measures such asvaccination, cytokine support (IL-2, IFNγ) or reducing immunosuppressantenvironment (ipilimumab) whilst the second seeks to restore an absolutedeficiency with components of a functional immune response (passiveimmunotherapy with antibodies, TCR transfer, Stem Cell Transplantationand adoptive immunotherapy). These approaches are unified by theargument that a highly effective functional anti-tumour immune responseis indeed possible. Although irrefutable evidence exists for aneffective anti-tumour immune response in some cases, this central pillarof tumour immunology is overwhelmingly countered by the current clinicalreality that despite great efforts, no effective immunotherapeutics areavailable for the majority of patients with cancer. Almost all cancervaccination trials have provided negative results, with those providingpositive data most frequently demonstrating a small effect. The realityis that therapeutic antibodies offer very modest clinical benefit in thearea of oncology. Thus, there remains a demand for more effectiveimmunotherapeutic agents.

Bispecific antibodies unify two antigen binding sites of differentspecificity into a single construct giving them the ability to bringtogether two discrete antigens with exquisite specificity. The firstbispecific antibodies resembled natural immunoglobulin G (IgG) moleculesin which the two arms were equipped with distinct binding specificities.The concept of using these bispecific antibodies to engage cytotoxic Tcells for cancer cell lysis was originally shown by Staerz andcolleagues in 1985 (Nature 1985, 314: 628). Since then, different kindsof constructs have been made and developed for the same purpose. Recentfocus has been on creating antibody constructs by joining two singlechain Fv regions (scFv antibody fragments) while omitting the Fc portionpresent in full immunoglobulins. Each scFv unit in such constructs(so-called bispecific T cell engagers or BITE antibodies) is made up ofone variable domain from each of the heavy (VH) and light (VL) antibodychains, joined with one another via a synthetic polypeptide linker. Theresulting bispecific single chain antibody is therefore a speciescontaining two VH/VL pairs of different specificity on a singlepolypeptide chain of approximately 55 kDa.

Promising experimental results have emerged from the use of BITEantibodies. Kufer and colleagues have demonstrated that CD3/targetantigen-bispecific antibodies of this design have an exceptionally highpotency, and can engage CD8+ and CD4+ T cells for redirected lysis ofcancer cells at very low effector to target ratios (Mack, PNAS (1995),92:7021-5). The antibodies have shown potential in treating tumours(Mack, J Immunol (1997), 158:3965-70; Kufer, Canc Immunol Immunother(1997), 45:193-7; and Loffler, Blood (2000), 95: 2098-103) and also intreating non-tumour diseases (Bruhl, J Immunol (2001), 166:2420-6). Theantibodies rely on one scFv unit being capable of activating T cells, byspecifically binding to an antigen on the T cells, and the other scFvunit specifically binding to an antigen on a target cell intended fordestruction. In this way, the antibodies are able to activate andredirect the immune system's cytotoxic potential for the destruction ofpathological cells.

Two BITE antibodies are currently being tested in clinical trials.Blinatumomab (also known as MT103) is bispecific for CD3 on T cells andCD19 on B cells, and is being tested for the treatment of non-Hodgkin'slymphoma and acute lymphoblastic leukaemia. MT110 is bispecific for CD3and epithelial cell adhesion molecule (EpCAM), and is being tested forthe treatment of lung and gastrointestinal cancer patients.

However, a limitation of BITE antibodies and similar bispecifictherapeutic antibodies is that they can activate T cells when binding toany target that expresses the targeting antigen. For instance, if theBITE or bispecific antibody was designed to target the EGF receptor,which is expressed at high levels in many epithelial cancers but alsoexpressed at moderate levels in healthy tissues, both healthy andcancers tissue are targeted. Moreover, the agent becomes active whenbound to any surface and thus non-specific binding to surfaces in anon-antigen binding manner can lead to T-cell activation and off-targeteffects. For these reasons, all BITE antibodies in development are givenat very low concentrations.

Accordingly, although bispecific antibody constructs have greattherapeutic value in redirecting the body's own immune system to achievethe eradication or neutralisation of unwanted cells, the activation ofsuch redirection requires that it be tightly controlled so that thecytotoxic potential is recruited and applied only in the directionintended. Thus, there is a strong need for further immunotherapeuticagents that overcome limitations described above.

The present inventors have now devised a means for recruiting andactivating immune cells (eg T cells) to target unwanted cells, whereinthe immune cells are only activated when in the vicinity of the targetedcells. As will become apparent below, such selective activation ofimmune cells is based on selective unmasking of an immune cell bindingregion in the vicinity of unwanted target cells by an agent that residesin the vicinity of the unwanted cells. In this way, the technology ismore specific for the unwanted cell (eg more cancer specific) by virtueof the immune cell binding region only being unmasked in the vicinity ofthe unwanted cell (eg in the presence of a cancer associated protease).This dramatically expands the therapeutic window of bispecificantibodies and so facilitates larger doses of drug to be given leadingto greater target effect.

Accordingly, a first aspect of the invention provides a moleculecomprising: (i) a targeting moiety capable of directly or indirectlytargeting to unwanted cells, and (ii) a further moiety that has a maskedimmune cell binding region so as to prevent binding of the furthermoiety to an immune cell, wherein the masked immune cell binding regionis capable of being selectively unmasked when the molecule is in thevicinity of the unwanted cells so as to allow binding of the furthermoiety to an immune cell. The targeting moiety functions to bring thefurther moiety into the vicinity of the unwanted cells, for example inthe same way as one of the antigen specificities of a BITE antibodytargets the construct to a cancer cell.

The selective unmasking of the immune cell binding region in thevicinity of the unwanted cells ensures that the further moiety can onlyengage immune cells when in the vicinity of the unwanted cells.

The individual components of the molecule are described in detail below.

Targeting Moiety

By ‘targeting moiety’, we include the meaning of any moiety that iscapable of targeting to the unwanted cells. Preferably, the targetingmoiety is capable of targeting selectively to the unwanted cells. Forexample, it is preferred if the targeting moiety targets unwanted cellsto a greater extent than it does normal cells, and most preferablytargets only unwanted cells.

It will be appreciated that binding of the targeting moiety to normalcells may be tolerated if they can be functionally replaced by othertherapeutic means or if they are not essential to life. Thus, atargeting moiety that targets to a cancer cell as well as, for example,an endocrine tissue or organ is not precluded. In this case, thetargeting moiety acts to redirect an immune response to both unwantedcells and to other cells that can be functionally replaced bytherapeutic means. In a life-saving situation for example, the tissue ororgan may be sacrificed provided its function was either not essentialto life, for instance in the case of the testes, prostate or pancreas,or could be supplied by hormone replacement therapy. Such considerationswould apply to the thyroid gland, parathyroids, adrenal cortex andovaries, for example.

It follows that the targeting moiety may be a moiety that is capable oftargeting selectively to unwanted cells as opposed to wanted cells,wherein the unwanted cells may include cells whose presence in a host isundesired and optionally cells whose presence in the host is desired butwhose presence can be functionally replaced by therapeutic means.

It is also appreciated that selective unmasking of the immune cellbinding regionconfers specificity on where the immune cell bindingregion is unmasked, and so binding of the targeting moiety to normalcells, in the vicinity of which the immune cell binding region is notunmasked, may also be tolerated.

Most preferably, however, the targeting moiety targets selectively tounwanted cells as opposed to any other cells.

In one embodiment, the targeting moiety is capable of directly targetingto unwanted cells. However, the targeting moiety may be one thatindirectly targets to unwanted cells, for example by targeting toanother moiety that is localised in the vicinity of the unwanted cells(e.g. by association with the unwanted cells), as described below.

Conveniently, the targeting moiety is a specific binding partner of anentity expressed by or associated with the unwanted cell. Typically, theexpressed entity is expressed selectively on the unwanted cell. Forexample, the abundance of the expressed entity is typically 10 or 100 or500 or 1000 or 5000 or 10000 higher on the unwanted cell than on othercells within the body to be treated. However, as mentioned above, theselective unmasking of the immune cell binding region in the vicinity ofthe unwanted cells provides additional specificity on where the immunecell binding region is unmasked and so the binding partner may bind anentity that is similarly or even underexpressed on unwanted cellsrelative to other cells within the body.

By “binding partner” we include the meaning of a molecule that binds toan entity expressed by a particular cell. Preferably, the bindingpartner binds selectively to that entity. For example, it is preferredif the binding partner has a K_(d) value (dissociation constant) whichis at least five or ten times lower (i.e. higher affinity) than for atleast one other entity expressed by another cell (e.g. a normal celltype), and preferably more than 100 or 500 times lower. More preferably,the binding partner of that entity has a K_(d) value more than 1000 or5000 times lower than for at least one other entity expressed by anothercell (e.g. normal cell type). K_(d) values can be determined readilyusing methods well known in the art. However, as discussed above, it isappreciated that the binding partner may bind selectively to an entityexpressed by an unwanted cell and by a normal cell provided that thenormal cell may be functionally replaced or else is not essential tolife. For example, in lymphoma, anti-CD20 (which targets all B cells) isvery effective and kills all B cells, healthy and malignant. However,this can be tolerated as B cells are not critical for health. Further,in the case of melanoma, lymphoma, prostate cancer, thyroid, testicularor ovarian cancer, targeting healthy counterpart tissue may also betolerated.

Typically, the binding partner is one that binds to an entity that ispresent or accessible to the binding partner in significantly greaterconcentrations in or on unwanted cells than in any normal cells of thehost. Thus, the binding partner may bind to a surface molecule orantigen on the unwanted cell that is expressed in considerably higheramounts than on normal cells. Similarly, the binding partner may bind toan entity that has been secreted into the extracellular fluid by theunwanted cells to a greater extent than by normal cells. For example,the binding partner may bind to a tumour associated antigen which isexpressed on the cell membrane or which has been secreted into tumourextracellular fluid.

The targeting moiety may be a polypeptide or a peptide. In aparticularly preferred embodiment, the targeting moiety is an antibodythat binds to an antigen expressed by the unwanted cell, for example atumour associated antigen.

Preferred antibody targets (with examples of unwanted cell types inparentheses) include: Her2/Neu (Epithelial malignancies); CD22 (B cells,autoimmune or malignant); EpCAM (CD326) (Epithelial malignancies); EGFR(epithelial malignancies); PMSA (Prostate Carcinoma); CD30 (B cellmalignancies); CD20 (B cells, autoimmune, allergic or malignant); CD33(Myeloid malignancies); membrane IgE (Allergic B cells); IgE Receptor(CD23) (Mast cells or B cells in allergic disease), CD80 (B cells,autoimmune, allergic or malignant); CD86 (B cells, autoimmune, allergicor malignant); CD2 (T cell or NK cell lymphomas); CA125 (multiplecancers including Ovarian carcinoma); Carbonic Anhydrase IX (multiplecancers including Renal Cell Carcinoma); CD70 (B cells, autoimmune,allergic or malignant); CD74 (B cells, autoimmune, allergic ormalignant); CD56 (T cell or NK cell lymphomas); CD40 (B cells,autoimmune, allergic or malignant); CD19 (B cells, autoimmune, allergicor malignant); c-met/HGFR (Gastrointestinal tract and hepaticmalignancies; TRAIL-R1 (multiple malignancies including ovarian andcolorectal carcinoma); DR5 (multiple malignancies including ovarian andcolorectal carcinoma); PD-1 (B cells, autoimmune, allergic ormalignant); PD1L (Multiple malignancies including epithelialadenocarcinoma); IGF-1R (Most malignancies including epithelialadenocarcinoma); VEGF-R2 (The vasculature associated with the majorityof malignancies including epithelial adenocarcinomas; Prostate stem cellantigen (PSCA) (Prostate Adenocarcinoma); MUC1 (Epithelialmalignancies); CanAg (tumors such as carcinomas of the colon andpancreas); Mesothelin (many tumours including mesothelioma and ovarianand pancreatic adenocarcinoma); P-cadherin (Epithelial malignancies,including breast adenocarcinoma); Myostatin (GDF8) (many tumoursincluding sarcoma and ovarian and pancreatic adenocarcinoma); Cripto(TDGF1) (Epithelial malignancies including colon, breast, lung, ovarian,and pancreatic cancers); ACVRL1/ALK1 (multiple malignancies includingleukaemias and lymphomas); MUC5AC (Epithelial malignancies, includingbreast adenocarcinoma); CEACAM (Epithelial malignancies, includingbreast adenocarcinoma); CD137 (B cells or T cells, autoimmune, allergicor malignant); CXCR4 (B cells or T cells, autoimmune, allergic ormalignant); Neuropilin 1 (Epithelial malignancies, including lungcancer); Glypicans (multiple cancers including liver, brain and breastcancers); HER3/EGFR (Epithelial malignancies); PDGFRa (Epithelialmalignancies); EphA2 (multiple cancers including neuroblastoma,melanoma, breast cancer, and small cell lung carcinoma); and CD138(Myeloma).

Particularly preferred targeting moiety antibodies include ananti-epidermal growth factor receptor antibody such as Cetuximab, ananti-Her2 antibody, an anti-CD20 antibody such as Rituximab, ananti-CD22 antibody such as Inotuzumab, an anti-CD70 antibody, ananti-CD33 antibody such as hp67.6 or Gemtuzumab, an anti-MUC1 antibodysuch as GP1.4 and SM3, an anti-CD40 antibody, an anti-CD74 antibody, ananti-P-cadherin antibody, an anti-EpCAM antibody, an anti-CD138antibody, an anti-E-cadherin antibody, an anti-CEA antibody, and ananti-FGFR3 antibody.

Yet further selective targets useful for preventing or treating variousconditions characterised by the presence of unwanted cells are providedbelow. For all of the examples below, therapeutic antibodies are alreadyavailable or can be readily prepared by the skilled person.

Target Unwanted cell Activin A Many types of carcinoma, lymphoma,sarcoma and leukaemia activin A, activin B and inhibin B Many types ofcarcinoma, lymphoma, sarcoma and leukaemia Adenocarcinoma antigen Manytypes of carcinoma, AFP (alpha-fetoprotein) Many types of carcinoma,amyloid beta (Abeta) Alzheimer's Disease amyloid beta (Abeta) peptideAβ40 Alzheimer's Disease amyloid beta (Abeta) peptide soluble monomerAlzheimer's Disease amyloid beta (Abeta) peptides Aβ42 and Aβ40Alzheimer's Disease ANGPT2 (Ang2, angiopoietin 2) Multiple carcinomasN-glycolyl GM3 ganglioside (N- Brain tumours glycolylneuraminic acid(NeuGc, NGNA) GM3 gangliosides, NeuGcGM3) Mus musculus IgM- kappa P3 BSG(basigin, Ok blood group, CD147) Many types of carcinoma, lymphoma,sarcoma and leukaemia CA 72-4 (tumour associated glycoprotein 72, Manytypes of carcinoma, TAG-72, TAG, HMW mucin-like glycoprotein) CA9(carbonic anhydrase IX, CAIX, MN, G250) Many types of carcinoma,carcinoma associated antigen CTAA16.88 Many types of carcinoma, (complexof cytokeratin polypeptides (35- 40 kDa)) CCL11 (chemokine (C-C motif)ligand 11, Many types of carcinoma and chemokine CC 11, eotaxin1)lymphoma/leukaemia CCL2 (chemokine (C-C motif) 2, chemokine Many typesof carcinoma and CC 2, monocyte chemoattractant protein-1,lymphoma/leukaemia MCP-1, monocyte chemotactic and activating factor,MCAF, small inducible cytokine A2, SCYA2, HC11) CCR4 (chemokine (C-Cmotif) receptor 4, Many types of carcinoma and chemokine CC receptor 4,CCR-4, CKR4, k5- lymphoma/leukaemia 5, CD194) CD14 Many types ofcarcinoma and lymphoma/leukaemia CD15 (3-fucosyl-N-acetyl-lactosamine,Lewis Many types of carcinoma and x, stage-specific embryonic antigen 1,SSEA-1) lymphoma/leukaemia CD19 (B lymphocyte surface antigen B4, Leu-Lymphoma and Acute lymphoblastic 12) leukaemia CD2 (lymphocytefunction-antigen 2, LFA-2) T-cell and NK-cell lymphoma CD200 (OX-2)T-cell and NK-cell lymphoma CD22 (sialic acid binding Ig-like lectin 2,Lymphoma and Acute lymphoblastic SIGLEC2, SIGLEC-2, B-lymphocyte cellleukaemia adhesion molecule, BL-CAM, Leu-14 CD33 (sialic acid bindingIg-like lectin 3, Myeloid leukaemia and Stem cells SIGLEC3, SIGLEC-3,gpG7, p67) CD38 (ADP-ribosyl cyclase 1, cyclic ADP- Myeloid leukaemiaand many types of ribose hydrolase 1, cADPr hydrolase 1, T10) carcinomaCD40 (tumor necrosis factor receptor Lymphoma and many types ofcarcinoma superfamily member 5, TNFRSF5, p50) CD40LG (CD40 ligand,CD40L, tumor necrosis Lymphoma and many types of carcinoma factor ligandsuperfamily member 5, TNFSF5, tumor necrosis factor related activationprotein, TRAP, CD154) CD44 (homing function and Indian blood groupMyeloid leukaemia and many types of system, chondroitin sulfateproteoglycan 8, carcinoma including cancer stem cells CSPG8) CD5 (T1,LEU-1) T-cell lymphoma, T-cells and B-cell lymphomas such as chroniclymphocytic leukaemia. CD52 T-cell lymphoma, T-cells and B-celllymphomas. Autoimmune induced immune cells may also be targeted. CD6(Tp120) T-cell lymphoma, T-cells and B-cell lymphomas such as chroniclymphocytic leukaemia. CD70 (tumor necrosis factor superfamily Lymphomaand many types of carcinoma member 7, TNFSF7, CD27LG, CD27L) CD74 (majorhistocompatibility class II Lymphoma and many types of carcinomainvariant chain, MH2) CD80 (B7-1, CD28LG1) Lymphoma and many types ofcarcinoma CD86 (B7-2, CD28LG2) Lymphoma and many types of carcinoma CEA(anticarcinoembryonic antigen) Many types of carcinoma, CEACAM3(carcinoembryonic antigen-related Many types of carcinoma, cell adhesionmolecule 3, CGM1, CD66d) CEACAM5 (carcinoembryonic antigen-related Manytypes of carcinoma, cell adhesion molecule 5, CEA, CD66e) CEACAM8(carcinoembryonic antigen-related Many types of carcinoma, cell adhesionmolecule 8, NCA-95, nonspecific cross-reacting antigen 95 kDa,granulocyte cell antigen, CGM6, CD66b) ClfA (Clumping factor A) Manytypes of carcinoma, complement C3b, C4b Many types of unwanted cells.CSF2 (colony stimulating factor 2 (granulocyte- Myeloid diseasesmacrophage), granulocyte-macrophage colony stimulating factor, GM-CSF)CSF2RA (colony-stimulating factor Myeloid diseases2(granulocyte-macrophage) receptor alpha subunit, GM-CSF-R-alpha, CD116)CSPG4 (chondroitin sulfate proteoglycan 4, Many types of carcinoma,lymphoma, sarcoma high molecular weight-melanoma-associated andleukaemia antigen, HMW-MAA) CTLA4 (cytotoxic T lymphocyte-associatedRegulatory T-cells and unwanted immune antigen 4, CD152) cells. ED-B(fibronectin extra domain B) Many types of carcinoma, EGFR (epidermalgrowth factor receptor, Many types of carcinoma, lymphoma, sarcomareceptor tyrosine-protein kinase erbB-1, and leukaemia ERBB1, HER1,HER-1, ERBB) EPCAM (epithelial cell adhesion molecule, Many types ofcarcinoma, lymphoma, sarcoma tumor-associated calcium signal transducer1, and leukaemia TACSTD1, gastrointestinal tumor-associated protein 2,GA733-2, epithelial glycoprotein 2, EGP-2, epithelial cell adhesionmolecule, Ep- CAM, KSA, KS1/4 antigen, M4S, tumor antigen 17-1A, EpCAM,CD326) ERBB2 (epidermal growth factor receptor 2, Many types ofcarcinoma, lymphoma, sarcoma receptor tyrosine-protein kinase erbB-2,and leukaemia EGFR2, HER2, HER-2, p185c-erbB2, NEU, CD340 ERBB3(receptor tyrosine-protein kinase erbB- Many types of carcinoma,lymphoma, sarcoma 3, HER3) and leukaemia FAP (fibroblast activationprotein, alpha) Many types of carcinoma, lymphoma, sarcoma andleukaemia. FCER2 (immunoglobulin E Fc receptor low Many types ofcarcinoma, lymphoma, sarcoma affinity II, Fc epsilon RII, CD23) andleukaemia in B-cells. FCGR1 (immunoglobulin G Fc receptor high Manytypes of carcinoma, lymphoma, sarcoma affinity I, Fc gamma RI, CD64,encoded by and leukaemia. human FCGR1A, FCGR1B, FCGR1C) fibrin II betachain (NH2 terminus) Many types of carcinoma, lymphoma, sarcoma andleukaemia. FLT1 (fms-related tyrosine kinase 1, vascular Many types ofcarcinoma, sarcoma, endothelial growth factor receptor 1, VEGFR-1,lymphoma, sarcoma and leukaemia. In VEGFR, FLT, FRT, vascularpermeability particular tumour blood vessels. factor receptor) FOLH1(folate hydrolase, prostate specific Many types of carcinoma, lymphoma,sarcoma membrane antigen, PSMA) and leukaemia in particular prostatecarcinoma and unwanted prostate tissue. FOLR1 (folate receptor 1, folatereceptor FR Many types of carcinoma, lymphoma, sarcoma alpha, FR-alpha,adult folate-binding protein, and leukaemia FBP, ovariantumor-associated antigen MOv18) GD2 ganglioside Brain tumours andunwanted neuronal tissue. GD3 ganglioside Brain tumours and unwantedneuronal tissue. GLP1R (glucagon-like peptide 1 receptor) Many types ofcarcinoma, lymphoma, sarcoma and leukaemia GPNMB (glycoproteintransmembrane NMB, Many types of carcinoma, lymphoma, sarcomahematopoeitic growth factor inducible and leukaemia neurokinin-1 type,HGFIN) hapten NP-cap (4-hydroxy-3-nitrophenacetyl Many types ofcarcinoma, lymphoma, sarcoma caproic acid) and leukaemia HAVCR1(hepatitis A virus cellular receptor 1, Hepatitis A infected cells.T-cell immunoglobulin and mucin domain- containing protein 1, TIM1,KIM-1) HBV (hepatitis B virus) HBV infected cells HCMV (humancytomegalovirus) gB CMV infected cells. glycoprotein HCV (hepatitis Cvirus) HCV infected cells heat shock protein 90 homolog Many types ofcarcinoma, lymphoma, sarcoma and leukaemia HGF (hepatocyte growthfactor, scatter factor, Hepatoma and hepatocellular carcinoma. Also SF,hepatopoeitin A) unwanted hepatic tissue. HIV-1 (human immunodeficiencyvirus) HIV infected cells HLA-DR10 (DRB1*1001) Autologous or AllogeneicMHC Class-II expressing cells including tumour cells HLA-DRB (HLA-DRbeta) Autologous or Allogeneic MHC Class-II expressing cells includingtumour cells HSV (herpes simplex virus) HSV infected cells ICAM1(intercellular adhesion molecule 1, Many types of carcinoma, lymphoma,sarcoma ICAM-1, CD54) and leukaemia ICAM3 (intercellular adhesionmolecule 3, Many types of carcinoma, lymphoma, sarcoma ICAM-3, CD50) andleukaemia Membrane Immunoglobulin IgE IgE secreting B-cells and Plasmacells (cuasing allergic disease). IgE Fc IgE secreting B-cells andPlasma cells (cuasing allergic disease). IGF1R (insulin-like growthfactor 1 receptor, Most types of carcinoma, lymphoma, sarcoma IGF1-R,IGF-1R, CD221) and leukaemia IGHE connecting region (CO) M1 prime (inIgE secreting cells such as B-cells and plasma alternatively splicedheavy chain of membrane cells. Particularly unwanted in allergicdisease. IgE on B cells IL2RA (interleukin-2 receptor, alpha subunit,B-cells and T-cells in either malignant or IL-2RA, TAC, CD25) autoimmunedisease. IL2RB (interleukin-2 receptor beta subunit, IL- B-cells andT-cells in either malignant or 2RB, p70, CD122) autoimmune disease.IL5RA (interleukin 5 receptor alpha subunit, B-cells and T-cells ineither malignant or CD125) autoimmune disease. IL6R (interleukin 6receptor, IL-6R, CD126) B-cells and T-cells in either malignant orautoimmune disease. ITGA2 {integrin alpha 2, GPla, subunit of the Manytypes of carcinoma, lymphoma, alpha2beta1 integrin (VLA-2, collagenleukaemias. receptor), CD49b) ITGA2B_ITGB3 (integrin alpha2b_beta3, Manytypes of carcinoma, lymphoma, integrin αllbβ3, GPllbllla, fibrinogenreceptor, leukaemias. CD41_CD61) ITGA4 (integrin alpha 4 subunit, CD49d)Many types of carcinoma, lymphoma, leukaemias. ITGA4_ITGB7 (integrinalpha4_beta7, integrin Many types of carcinoma, lymphoma, α4β7,lymphocyte Peyer's patch adhesion leukaemias. molecule 1, LPAM-1) ITGA5(integrin alpha 5 subunit, CD49e) Many types of carcinoma, lymphoma,leukaemias. ITGAE_ITGB7 (integrin alphaE_beta7, integrin Many types ofcarcinoma, lymphoma, αEβ7, human mucosal lymphocyte antigen 1,leukaemias. HML-1) ITGAL (integrin alpha L subunit, lymphocyte Manytypes of carcinoma, lymphoma, function associated antigen 1, CD11a)leukaemias. ITGAV_ITGB3 (integrin alphaV_beta3, integrin Many types ofcarcinoma, lymphoma, aVβ3, CD51_GPllla, vitronectin receptor, VNR,leukaemias. CD51_CD61 ITGB1 (integrin beta1 subunit, GPlla, CD29) Manytypes of carcinoma, lymphoma, leukaemias. ITGB2 (integrin beta2 subunit,LFA-1, MAC-1, Many types of carcinoma, lymphoma, CD18) leukaemias. KDR(kinase insert domain receptor, vascular Many types of carcinoma,lymphoma, endothelial growth factor receptor 2, VEGFR2, leukaemias.VEGF-R2, FLK1, CD309) LTA (lymphotoxin alpha, TNF superfamily Many typesof carcinoma, lymphoma, member 1, TNFSF1, LT) leukaemias. LTB(lymphotoxin beta, TNF superfamily Many types of carcinoma, lymphoma,member 3, TNFSF3, p33) leukaemias. MET (met proto-oncogene, hepatocytegrowth Many types of carcinoma, lymphoma, factor HGF receptor, HGFR,scatter factor SF leukaemias. receptor, HGF/SF receptor, tyrosineprotein kinase c-met, papillary renal cell carcinoma 2, RCCP2) MS4A1(membrane-spanning 4-domains Many types of carcinoma, lymphoma,subfamily A member 1, CD20) leukaemias. MSLN (mesothelin,pre-pro-megakaryocyte- Many types of carcinoma, lymphoma, potentiatingfactor, megakaryocyte potentiating leukaemias. factor, MPF, CAK1) MST1R(macrophage stimulating 1 receptor, Many types of carcinoma, lymphoma,macrophage stimulating protein receptor, MSP leukaemias. receptor,c-met-related tyrosine kinase, protein-tyrosine kinase 8, PTK8, RON,p185- Ron, CD136) MSTN (myostatin, growth differentiation factor Manytypes of carcinoma, lymphoma, 8, GDF8) leukaemias. MUC1 (mucin 1,polymorphic epithelial mucin, Many types of carcinoma, lymphoma, PEM,episialin, CD227) leukaemias. MUC1 sialylated carbohydrate, tumour- Manytypes of carcinoma, lymphoma, associated (CA242, cancer antigen 242)leukaemias. MUC16 (mucin 16, MUC-16, cancer antigen Many types ofcarcinoma, lymphoma, 125, CA125) leukaemias. MUC5AC (mucin 5AC, mucin 5subtypes A and Many types of carcinoma, lymphoma, Ctracheobronchial/gastric) leukaemias. N-glycolyl GM3 ganglioside (N-Brain tumours and unwanted neural tissue. glycolylneuraminic acid(NeuGc, NGNA) GM3 ganglioside, NeuGcGM3) NCA-90 (nonspecificcross-reacting antigens Many types of carcinoma, lymphoma, sarcoma 90kDa glycoproteins, granulocyte cell antigen) and leukaemia NCAM1 (neuralcell adhesion molecule 1, Brain tumours and unwanted neural tissue alsoNCAM-1, NCAM, CD56) many types of carcinoma and lymphoma. Nectin-4 Manytypes of carcinoma, lymphoma, sarcoma and leukaemia NGF (nerve growthfactor, nerve growth factor Many types of carcinoma, lymphoma, sarcomabeta polypeptide, NGFB, beta-NGF) and leukaemia NIP-cap(3-iodo-4-hydroxy-5-nitrophenyl- Many types of carcinoma, lymphoma,sarcoma acetyl caproic acid) and leukaemia NRP1 (neuropilin 1, NRP,vascular endothelial Many types of carcinoma, lymphoma, sarcoma cellgrowth factor 165 receptor, VEGF165 and leukaemia receptor, VEGF165R,CD304) PDGFRA (platelet-derived growth factor Many types of carcinoma,lymphoma, sarcoma receptor alpha subunit, PDGFR2, CD140a) and leukaemiaphosphatidylserine Many types of carcinoma, lymphoma, sarcoma andleukaemia particularly apoptotic cells. PSCA (prostate stem cellantigen) Many types of carcinoma and leukaemia. RSV (human respiratorysyncytial virus, RSV infected cells glycoprotein F) RTN4 (reticulon 4,neurite outgrowth inhibitor, Many types of carcinoma, lymphoma, sarcomaNOGO) and leukaemia SDC1 (syndecan-1, CD138) Unwanted plasma cells foundin plasma cell dyscrasias, particularly Myeloma, Amyloidosis and MGUS.SELE (E-selectin, CD62E) Many types of carcinoma and lymphoma. SELL(L-selectin, CD62) Many types of carcinoma and lymphoma. SELP(P-selectin, CD62) Many types of carcinoma and lymphoma. SFRP1 (selectedfrizzled-related protein 1, Many types of carcinoma and lymphoma. fusionregulatory protein 1, FRP-1) SLAMF7 (SLAM family member 7, CD2 subsetMany types of unwanted cells including tumour 1, CS1, CD2-likereceptor-activating cytotoxic cells and those involved in autoimmunecells, CRACC, 19A24, CD319) disease. SLC3A2 (solute carrier family 3(activators of Many types of unwanted cells including tumour dibasic andneutral amino acid transport) cells and those involved in inflammatorymember 2, 4F2 antigen heavy chain, 4F2HC, disease. CD98 heavy chain,CD98hc, CD98) SOST (sclerostin) Bone disease including oesteosarcoma andosteoporosis. Staphylococcus epidermidis lipoteichoic acidStaphylococcus infected tissue. T cell receptor (TR) TR alpha_betaT-cell lymphoma or autoimmune-causing T- cells. TGFB1 (transforminggrowth factor beta1, TGF Many types of unwanted cells including tumourbeta) cells and those involved in fibrotic disease. TGFB2 (transforminggrowth factor beta 2) Many types of unwanted cells including tumourcells and those involved in fibrotic disease. TNF (tumor necrosis factor(TNF) superfamily Many types of unwanted cells including tumour member2, TNFSF2, TNF-alpha, TNFA) cells and those involved in inflammatorydisease. TNFRSF10A (tumor necrosis factor receptor Many types ofunwanted cells including tumour (TNFR) superfamily member 10A, deathcells and those involved in inflammatory receptor 4, DR4, TNF-relatedapoptosis- disease. inducing ligand receptor 1, TRAILR1, TRAIL- R1,TR-1, CD261) TNFRSF10B (tumor necrosis factor receptor Many types ofunwanted cells including tumour (TNFR) superfamily member 10B, deathcells and those involved in inflammatory receptor 5, DR5, TNF-relatedapoptosis- disease. inducing ligand receptor 2, TRAILR2, TRAIL- R2,TR-2, CD262) TNFRSF12A (tumor necrosis factor receptor Many types ofunwanted cells including tumour (TNFR) superfamily member 12A,fibroblast cells and those involved in inflammatory growth factor(FGF)-inducible 14 kDa protein, disease. Fn14, TNF-like weak inducer ofapoptosis (Tweak) receptor, Tweak receptor, TweakR, CD266 TNFRSF8 (tumornecrosis factor receptor Many types of unwanted cells including tumour(TNFR) superfamily member 8, CD30) cells (in particular lymphoma) andthose involved in inflammatory disease. TNFRSF9 (tumor necrosis factorreceptor Many types of unwanted cells including tumour (TNFR)superfamily member 9, 4-1BB, T cell cells and those involved ininflammatory and antigen ILA, CD137 autoimmune disease. TNFSF11 (tumornecrosis factor (TNF) Many types of unwanted cells including tumoursuperfamily member 11, osteoclast cells and those involved ininflammatory and differentiation factor, ODF, OPGL, RANKL, autoimmunedisease. TRANCE, CD254) TNFSF13 (tumor necrosis factor (TNF) Many typesof unwanted cells including tumour superfamily member 13, aproliferation- cells and those involved in inflammatory and includingligand, APRIL, CD256 autoimmune disease. TNFSF13B (tumor necrosis factor(TNF) Many types of unwanted cells including tumour superfamily member13B, B cell activating cells and those involved in inflammatory andfactor, BAFF, TALL1, BLyS, B lymphocyte autoimmune disease. activator,CD257) TNFSF14 (tumor necrosis factor (TNF) Many types of unwanted cellsincluding tumour superfamily member 14, LIGHT, HVEM-L, cells and thoseinvolved in inflammatory and CD258) autoimmune disease. TNFSF4 (tumornecrosis factor (TNF) Many types of unwanted cells including tumoursuperfamily member 4, OX40 ligand, OX-40L, cells and those involved ininflammatory and TAX transcriptionally-activated glycoprotein 1,autoimmune disease. TXGP1, gp34, CD252) TPBG (trophoblast glycoprotein,5T4) Multiple carcinomas. TYRP1 (tyrosinase-related protein 1, 5,6-Multiple carcinomas. dihydroxyindole-2-carboxylic acid oxidase, DHICAoxidase, TRP1, melanoma antigen gp75) VAP-1 (vascular adhesion protein)Multiple carcinomas and hepatomas. VEGFA (vascular endothelial growthfactor A, Multiple carcinomas and hepatomas. VEGF-A, VEGF) VIM(vimentin) Multiple carcinomas and hepatomas.

Examples of tumour-associated, immune cell-associated and infectionreagent-related antigens which may be targeted by the targeting moietyare given in Table 1.

TABLE 1 Cell surface antigens for targeting Antigen Antibody Existinguses a) Tumour Associated Antigens Carcino-embryonic C46 (Amersham)Imaging and therapy of Antigen 85A12 (Unipath) colon/rectum tumours.Placental Alkaline H17E2 (ICRF, Imaging and therapy of PhosphataseTravers & Bodmer) testicular and ovarian cancers. Pan Carcinoma NR-LU-10(NeoRx Imaging and therapy of Corporation) various carcinomas includingsmall cell lung cancer. Polymorphic Epithelial HMFG1 (Taylor- Imagingand therapy of Mucin (Human milk Papadimitriou, ovarian cancer and fatglobule) ICRF) pleural effusions. β-human Chorionic W14 Targeting ofGonadotropin carboxypeptidase to human xenograft choriocarcinoma in nudemice (Searle et al (1981) Br. J. Cancer 44, 137-144). A carbohydrate onL6 (IgG2a)¹ Targeting of alkaline Human Carcinomas phosphatase (Senteret al (1988) PNAS USA 85, 4842-4846. CD20 Antigen on B 1F5 (IgG2a)²Targeting of alkaline Lymphoma (normal phosphatase (Senter et andneoplastic) al (1988) PNAS USA 85, 4842-4846. b) Immune Cell AntigensPan T Lymphocyte OKT-3 (Ortho) As anti-rejection Surface Antigen (CD3)therapy for kidney transplants. B-lymphocyte Surface RFB4 (Janossy,Immunotoxin therapy of Antigen (CD22) Royal Free Hospital) B celllymphoma. Pan T lymphocyte H65 (Bodmer and Immunotoxin treatment SurfaceAntigen (CD5) Knowles, ICRF; of acute graft versus licensed to Xoma hostdisease, Corp., USA) rheumatoid arthritis. c) Infectious Agent-RelatedAntigens Mumps virus-related Anti-mumps Antibody conjugated topolyclonal antibody diphtheria toxin for treatment of mumps. Hepatitis BSurface Anti HBs Ag Immunotoxin against Antigen hepatoma. ¹Hellström etal (1986) Cancer Res. 46, 3917-3923 ²Clarke et al (1985) Proc. Natl.Acad. Sci. USA 82, 1766-1770 Other antigens include alphafoetoprotein,Ca-125 and prostate specific antigen.

As used herein, the term “antibody” includes but is not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, fragmentsproduced by a Fab expression library and bispecific antibodies. Suchfragments include fragments of whole antibodies which retain theirbinding activity for a target substance, Fv, F(ab′) and F(ab′)2fragments, as well as single chain antibodies (scFv), fusion proteinsand other synthetic proteins which comprise the antigen-binding site ofthe antibody. A targeting moiety comprising only part of an antibody maybe advantageous by virtue of optimising the rate of clearance from theblood and may be less likely to undergo non-specific binding due to theFc part. Also included are domain antibodies (dAbs), diabodies, camelidantibodies and engineered camelid antibodies. Furthermore, foradministration to humans, the antibodies and fragments thereof may behumanised antibodies, which are now well known in the art (Janeway et al(2001) Immunobiology., 5th ed., Garland Publishing); An et al (2009)Therapeutic Monoclonal Antibodies: From Bench to Clinic, ISBN:978-0-470-11791-0).

Also included are asymmetric IgG-like antibodies (eg triomab/quadroma,Trion Pharma/Fresenius Biotech; knobs-into-holes, Genentech; Cross MAbs,Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech;strand exchange engineered domain (SEED) body, EMD Serono; biolonic,Merus; and Fab-exchanged antibodies, Genmab), symmetric IgG-likeantibodies (eg dual targeting (DT)-Ig, GSK/Domantis; two-in-oneantibody, Genentech; crosslinked MAbs, karmanos cancer center; mAb²,F-star; and Cov X-body, Cov X/Pfizer), IgG fusions (eg dual variabledomain (DVD)-Ig, Abbott; IgG-like bispecific antibodies, Eli Lilly;Ts2Ab, Medimmune/AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idec; TvAb,Roche) Fc fusions (eg ScFv/Fc fusions, Academic Institution; SCORPION,Emergent BioSolutions/Trubion, ZymoGenetics/BMS; dual affinityretargeting technology (Fc-DART), MacroGenics; dual (ScFv)₂-Fab,National Research Center for Antibody Medicine) Fab fusions (eg F(ab)₂,Medarex/AMGEN; dual-action or Bis-Fab, Genentech; Dock-and-Lock (DNL),ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv,UCB-Celltech), ScFv- and diabody-based antibodies (eg bispecific T cellengagers (BiTEs), Micromet; tandem diabodies (Tandab), Affimed; DARTs,MacroGenics; Single-chain diabody, Academic; TCR-like antibodies, AIT,Receptor Logics; human serum albumin ScFv fusion, Merrimack; andCOMBODIES, Epigen Biotech), IgG/non-IgG fusions (eg immunocytokins,EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantigen fusionprotein, Active Biotech; and immune mobilising mTCR Against Cancer,ImmTAC) and oligoclonal antibodies (eg Symphogen and Merus).

The antibody may possess any of the antibody-like scaffolds described byCarter (2006) “Potent antibody therapeutics by design”, Nat Rev Immunol.6(5):343-57, and Carter (2011) “Introduction to current and futureprotein therapeutics: a protein engineering perspective”, Exp Cell Res.317(9): 1261-9. incorporated herein by reference, together with thespecificity determining regions described herein. Thus, the term“antibody” also includes affibodies and non-immunoglobulin basedframeworks.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFvand dAb antibody fragments can be expressed in and secreted from E. colior yeast, thus allowing convenient production in the laboratory andeconomical production on a commercial scale.

The antibody may be of any of the IgG, IgE, IgA, IgM and IgD classes andmay be derived from any species. If the antibody is an IgG, it may beany of IgG1, IgG2, IgG3 or IgG4. It is preferred, however, that when theagent is for administration to a particular host, that the antibody, orat least the constant regions thereof, are derived from that host. Forexample, when the agent is to be administered to a human, the antibodyis preferably a human antibody or a humanized antibody, and so on.

Suitable antibodies that bind to particular antigens expressed byunwanted cells can be made by the skilled person using technologylong-established in the art. Methods of preparation of monoclonalantibodies and antibody fragments are well known in the art and includehybridoma technology (Kohler & Milstein (1975) “Continuous cultures offused cells secreting antibody of predefined specificity. Nature 256:495-497); antibody phage display (Winter et al (1994) “Making antibodiesby phage display technology.” Annu. Rev. Immunol. 12: 433-455); ribosomedisplay (Schaffitzel et al (1999) “Ribosome display: an in vitro methodfor selection and evolution of antibodies from libraries.” J. Immunol.Methods 231: 119-135); and iterative colony filter screening (Giovannoniet al (2001) “Isolation of anti-angiogenesis antibodies from a largecombinatorial repertoire by colony filter screening.” Nucleic Acids Res.29: E27). Further, antibodies and antibody fragments suitable for use inthe present invention are described, for example, in the followingpublications: “Monoclonal Hybridoma Antibodies: Techniques andApplication”, Hurrell (CRC Press, 1982); “Monoclonal Antibodies: AManual of Techniques”, H. Zola, CRC Press, 1987, ISBN: 0-84936-476-0;“Antibodies: A Laboratory Manual” 1^(st) Edition, Harlow & Lane, Eds,Cold Spring Harbor Laboratory Press, New York, 1988. ISBN 0-87969-314-2;“Using Antibodies: A Laboratory Manual” 2^(nd) Edition, Harlow & Lane,Eds, Cold Spring Harbor Laboratory Press, New York, 1999. ISBN0-87969-543-9; and “Handbook of Therapeutic Antibodies” Stefan Dübel,Ed., 1^(st) Edition,—Wiley-VCH, Weinheim, 2007. ISBN: 3-527-31453-9.

It is especially preferred if the targeting moiety is a single chainantibody such as a scFv antibody that comprises a heavy chain variabledomain and a light chain variable domain, which domains are joinedtogether by a flexible peptide linker. Preferably, the single chainantibody binds to an entity expressed by the unwanted cell, includingany of those mentioned above. However, it will also be understood thatthe targeting moiety may comprise only one antibody variable domain,such as a heavy chain variable domain or a light chain variable domain,and such a domain may bind to an entity expressed by the unwanted cell.

It will be appreciated that the targeting moiety may also be anycompound or part thereof that specifically binds, in a non-immune sense,to an entity expressed by unwanted cells or otherwise becomes associatedwith the unwanted cells. Thus, the specific binding partner may be anyof a hormone, a growth factor, a cytokine, or a receptor ligand (e.g.agonist or antagonist).

For example, cytokines have previously been used to target toxins toinvading bacterial. Using genetic engineering, recombinant proteins havebeen produced which contain for example IL-2 and a bindingdomain-deleted Pseudomonas exotoxin protein (Lorderboum-Galski et al,1988 (62)). This immunotoxin was effective in experimental animal models(Kozak et al, 1990 (63)). Fusion proteins have also been produced withIL-4, IL-6, alpha-MSH, EGF and TNF-alpha (reviewed in Waldmann 1992(35)), all of which are appropriate for use as targeting moieties in thepresent invention.

Particularly useful targeting moieties include cytokines such as IL-2,EGF, VEGF, Flt3L, HGF, IGF, IL-6, or IL-4. IL-2 and IL-4 can target toadult T cell leukaemia/lymphoma cells which express the high affinityIL-2 receptor whereas normal resting T-cells do not, or to T-cellsexpressing the IL-4 receptor. It has previously been shown that themonoclonal antibody MR6, which binds to the human IL-4 receptor, caninhibit the IL-4 induced proliferation of cloned helper T cells and theproduction of IgE by polyclonal B cells (Larche et al, 1988 (36)). Suchtargeting moieties may be used to eliminate a lymphoid cellsubpopulation in autoimmune disease or allergy.

Insulin like growth factors (IGF-1 and IGF-11) are preferentially takenup by malignant cells and so may be used to target tumour cells.Similarly EGF can be used to target malignant cells which upregulate theEGF receptor. Also, tumour associated blood vessels overexpress VEGFreceptor and so can be targeted by the family of VEGF growth factors.

Flt3 receptor is overexpressed in leukaemias and may be a therapeutictarget for acute and chronic leukaemias and myeloproliferativedisorders.

Myeloma cells express IL-6 receptor and also secrete IL-6 which acts inan autocrine fashion to stimulate cell proliferation. Thus IL-6 may beused as a targeting moiety for myeloma.

In another example, the targeting moiety is melanoma stimulating hormone(MSH) which binds to the MSH receptor which is expressed in high numbersin melanoma cells.

It is appreciated that a person skilled in the art can readily selectsuitable binding partners for any given unwanted cell, for example byidentifying surface antigens or molecules specific for that unwantedcell and finding a binding partner for that antigen or molecule.Considerable research has been carried out on antibodies and fragmentsthereof to tumour-associated antigens, immune cell antigens andinfectious agents, as described above. Thus, conveniently, selecting anappropriate targeting moiety for a given cell type typically involvessearching the literature. Alternatively, an unwanted cell is taken froma patient (e.g. by biopsy), and antibodies directed against the cellprepared. Such ‘tailor-made’ antibodies are already known. It has beendemonstrated that antibodies confer binding to tumour cells not onlyfrom the patient they have been obtained from but also for a largenumber of other patients. Thus, a plurality of such antibodies hasbecome commercially available. Other methods of identifying suitablebinding partners for a given unwanted cell include genetic approaches(eg microarray), proteomic approaches (eg differential Massspectrometry), immunological approaches (eg immunising animals withtumour cells and identifying antibody-secreting clones whichspecifically target malignant cells) and in silico approaches whereintargets are identified using a systems biology approach.

Further selective targets and suitable binding partners are shown inTable 2.

TABLE 2 Binding partners for tumour- selective targets andtumour-associated antigens Target Binding Partner Disease Truncated EGFRanti-EGFR mAb Gliomas Idiotypes anti-id mAbs B-cell lymphomas EGFR(c-erbB1) EGF, TGFα anti-EGFR Breast cancer mAb c-erbB2 mAbs Breastcancer IL-2 receptor IL-2 Lymphomas and anti-Tac mAb leukaemias IL-4receptor IL-4 Lymphomas and leukaemias IL-6 receptor IL-6 Lymphomas andleukaemias MSH (melanocyte- α-MSH Melanomas stimulating hormone)receptor Transferrin receptor Transferrin anti-TR Gliomas (TR) mAbgp95/gp97 mAbs Melanomas p-glycoprotein cells mAbs drug-resistantcluster-1 antigen (N- mAbs Small cell lung CAM) carcinomas cluster-w4Abs Small cell lung carcinomas cluster-5A mAbs Small cell lungcarcinomas cluster-6 (LeY) mAbs Small cell lung carcinomas PLAP(placental mAbs Some seminomas alkaline phosphatase) Some ovarian; somenon small cell lung cancer CA-125 mAbs Lung, ovarian ESA (epithelialspecific mAbs carcinoma antigen) CD 19, 22, 37 mAbs B-cell lymphomas 250kDa mAbs Melanoma proteoglycan p55 mAbs Breast cancer TCR-IgH fusionmAbs Childhood T-cell leukaemia Blood gp A antigen (in mAbs Gastric andcolon B or O individuals) tumours Mucin protein core mAbs Breast cancer

Further targets useful in preventing or treating various cancers areprovided below.

Target Cancer EpCam Bladder PMSA Prostate EGFR Breast Lung GlioblastomaColon CD20 Lymphoma CD22 Lymphoma CD52 Lymphoma Leukaemia

As an alternative to the targeting moiety being a specific bindingpartner, the targeting moiety may be a non-specific molecule that iscapable, following administration to a subject, of accumulating in thevicinity of the unwanted cells. For example, it is known thatmacromolecules accumulate non-specifically in tumours. Macromoleculesknown to accumulate in tumours non-specifically include albumin,immunoglobulins, transferrin, liposomes, nanoparticles (eg colloidalnanoparticles) and biodegradable polymers including dextrans,polyethylene glycol, polylysine and hydroxypropylmethylacrylamide.Macromolecules accumulate in human xenografted tumours in nude mice upto about 2.0% of administered dose per gram of tumour. Macromoleculessuch as polyethylene glycol and dextrans have been found to modify theclearance rate of substances to which they are attached and modify theirconcentration in tumours (Melton et al, 1987; Eno-Ammoquaye et al,1996). In exceptional tumours, a non-specific macromolecule mayaccumulate in greater concentration than an antibody directed at thesecreted antigen (Searle et al, 1981).

The discovery that such macromolecules accumulate in tumours has beencalled the Enhanced Permeability and Retention (EPR) effect, and hasbeen attributed to the leakiness of tumour capillaries and deficientlymphatic drainage (Matsumura & Macda, 1986).

Thus, when the unwanted cells are tumour cells, the targeting moiety maybe any of these macromolecules which accumulate in tumours. Preferably,the macromolecule used in the invention is hydrophilic and ischaracterised by being soluble in body fluids and in conventional fluidsfor parenteral administration. Suitably, the macromolecule isbiodegradable so that systemic accumulation during repeatedadministration is avoided. Clearly, however, it must not be degraded sofast as to fail to accumulate at the site of the unwanted cells (e.g.tumour). Preferably, the molecular weight and size of the agentcomprising such a macromolecule targeting moiety exceeds that of therenal threshold for urinary excretion (MW 60 000), as this helps theblood concentration to be sufficient to provide an effectiveblood:tumour concentration gradient. A molecular weight of up to atleast 800 000 is generally suitable, for example up to 160 000. Themacromolecule is preferably one which is not readily captured by thereticuloendothelial system. The molecular weights given exclude anywater of hydration.

Macromolecules that are available as sub-units and are not biodegradablemay be linked by biodegradable linking units so that thenon-biodegradable components are filtered through the kidneys andexcreted in the urine.

Alternatively, it is preferred if the polymer used to make themacromolecule is not biodegradable such that the molecular weight of anynon-biodegradable portion of the conjugate should be less than the renalthreshold (circa 70000) so that after degradation of the biodegradableportion the residual non-biodegradeable portion is excreted through thekidneys.

Conveniently, the macromolecule may be any of a dextran; a polyaminoacid; a nanoparticle (eg colloidal nanoparticle), or anon-tumour-specific protein such as an immunoglobulin, an albumin or atransferrin. Suitably, it may be a copolymer of styrene and maleicanhydride, or may be polyaspartic acid, poly-L-lysine, polyethyleneimineor polyethylene glycol.

It is appreciated that such macromolecules are used inmelanocyte-directed enzyme prodrug therapy (MDEPT), as described in WO1998/024478.

In addition to directly targeting an unwanted cell, the targeting moietymay be one that is capable of indirectly targeting to unwanted cells bybeing capable of binding to a moiety that is capable of targeting tounwanted cells. For example, a moiety that is capable of directlytargeting to an unwanted cell (including any of those described above)may comprise a first binding partner. A targeting moiety that comprisesa second binding partner, capable of binding to the first bindingpartner, then has the ability to indirectly target the unwanted cells byvirtue of it being able to bind to the first binding partner. In thiscase, the targeting moiety does not bind to an entity expressed by orassociated with the unwanted cell directly, however it does soindirectly by binding to a moiety that does bind to an entity expressedby or associated with the unwanted cell. It will be understood that themolecule of the invention may comprise targeting moieties thatindirectly target unwanted cells in this way, as well as those targetingmoieties that target unwanted cells directly. By the first and secondbinding partners, we include the meaning of any two moieties which bindto each other selectively. Most preferably, the first and second bindingpartners only bind to each other and not to any other moieties.Non-covalent binding such as between biotin/avidin or streptavidin, orimmunological bindings are preferred. Thus, the first binding partnermay be biotin and the second binding partner may be avidin, and viceversa. Alternatively, the first binding partner may be an antigen andthe second binding partner may be an antibody specific for that antigen,and vice versa. However, any pair of first and second binding partnersthat selectively bind to each other may be used, and suitable pairs willbe known to the skilled person.

Unwanted Cell

The unwanted cell may be any cell whose presence in a host is undesired.Thus, the cell may be a tumour cell (benign or malignant), a cell from atumour microenvironment such as tumour fibroblasts or tumour bloodvessels, a virally infected cell, a cell introduced as part of genetherapy, or a normal cell which one wishes to destroy for a particularreason. For instance, it may be desirable to eliminate a subpopulationof immune cells such as T lymphocytes in autoimmune disease or such as Blymphocytes in allergic disease.

Preferably, the unwanted cell is one whose presence characterises acondition in a patient. By a condition characterised by the presence ofunwanted cells we include any biological or medical condition ordisorder in which at least part of the pathology is mediated by thepresence of unwanted cells. The condition may be caused by the presenceof the unwanted cells or else the presence of the unwanted cells may bean effect of the condition. Examples of particular conditions includetumours (benign or malignant), autoimmune conditions, cardiovasculardiseases, degenerative diseases, diabetes, allergic disease (eg asthma),neurodegenerative diseases such as Alzheimer's, transplantation patientsand infectious diseases.

For autoimmune disease, the unwanted cells may represent cells of theadaptive or innate immune response, preferably T cells, but morepreferably B cells. For cardiovascular disease, the unwanted cells mayrepresent cells within atheromatous lesions such as macrophages. Fordegenerative diseases, the unwanted cells may represent cells whichinduce the neurodegenerative changes, for instance in Alzheimer'sdisease they may be microglia or astrocytes. For other degenerativediseases any cell which facilitates the process of degeneration orapoptosis may be considered a target. For processes such as aging whereunwanted tissue builds up, for example in benign prostatic hyperplasis,non-malignant prostatic tissue would be a preferred target. For allergicdisease, cells which participate in the allergic reaction such as tissuemast cells may be considered an ideal target, but also IgE secretingcells such as plasma cells or B cells. In transplantation, alloreactivelymphocytes would represent a preferred target cell. In the context ofinfectious disease, any cell harbouring a virus, bacteria or fungalpathogen may be considered a preferred target cell for example an HIVinfected cell.

Immune Cell Binding Region of Further Moiety

By “immune cell binding region” we include the meaning of a region ofthe further moiety that is capable of binding to an immune cell.Preferably, the immune cell binding region is capable of selectivelybinding to an immune cell. For example, it is preferred if the immunecell binding region binds to an immune cell to a greater extent than itdoes any other type of cell, and most preferably binds to an immune cellonly.

Generally, the immune cell binding region binds to an entity expressedby an immune cell, and preferably binds selectively to that entity.Thus, the further moiety may be a binding partner for an entityexpressed on an immune cell wherein the immune cell binding region isthe region of the further moiety that is responsible for binding. It ispreferred if the immune cell binding region has a K_(d) value forbinding to an entity expressed by a given immune cell which is at leastfive or ten times lower than for at least one other entity expressed byanother cell type, and preferably more than 100 or 500 times lower. Morepreferably, the immune cell binding region has a K_(d) value for bindingto an entity expressed by a given immune cell which is at least 1000 or5000 times lower than for at least one other entity expressed by anothercell type.

By ‘immune cell’, we include the meaning of any immune cell within thenatural repertoire of cells in the immune system (e.g. human immunesystem) which, when activated, is able to bring about a change in theviability of a target cell. By ‘viability of a target cell’ we includethe meaning of the target cell's ability to survive, proliferate and/orinteract with other cells. Such interaction may either be direct, forexample when the target cell contacts another cell, or indirect, forexample when the target cell secretes substances which have an influenceon the functioning of another distant cell. Generally, the immune cellis one that reduces one or more of the target cell's ability to survive,proliferate and/or interact with other cells, and preferably, the immunecell is one that kills the target cell. It will be appreciated thereforethat the immune cell is preferably an immune effector cell.

In an embodiment, the immune cell is a member of the lymphoid celllineage, and so may be any of a T cell or a natural killer (NK) cell.Advantageously, such cells will have a cytotoxic or an apoptotic effecton the unwanted cell.

In another embodiment, the immune cell is a member of the myeloidlineage, and so may be any of a mononuclear phagocyte (eg monocyte ormacrophage), a neutrophilic granulocyte or a dendritic cell.Advantageously, such cells will have a cytotoxic, phagocytic or anapoptotic effect on the unwanted cell.

It is especially preferred if the immune cell is a T cell (eg cytotoxicT cell), and so the immune cell binding region of the further moiety isa T cell binding region. Thus, the invention provides a moleculecomprising: (i) a targeting moiety capable of directly or indirectlytargeting to unwanted cells, and (ii) a further moiety that has a maskedT cell binding region so as to prevent binding of the further moiety toa T cell, wherein the masked immune cell binding region is capable ofbeing selectively unmasked when the molecule is in the vicinity of theunwanted cells so as to allow binding of the further moiety to an immunecell.

By ‘T cell’, we include all types of T cell including CD4+, CD8+, γδ Tcells and NK-T cells. Advantageously, the T cell is a cytotoxic T cellsuch that a cytotoxic T cell response is recruited. In this way, acytotoxic effect can be exerted on the unwanted cells.

Entities expressed on the surface of T cells include any of CD3, T cellreceptor (TCR), CD4, CD8 and CD28, and so when immune cell bindingregion is one that binds to T cells, the further moiety may be capableof binding to any of these antigens. For example, the further moiety maybe an antibody that binds specifically to any of CD3, TCR, CD4, CD8 andCD28.

Entities expressed on the surface of other immune cells are well knownin the art and, as with those on the surface of T cells, can be readilyidentified by interrogating the scientific literature. Examples includeCD16, CD56, CD8, NK cell receptor (eg CD94:NKG2 heterodimer, Ly49homodimer. γδ T cell receptor, pathogen recognition receptor, stresssurveillance receptor, Killer-cell Ig-like receptor (KIR) or leukocyteinhibitory receptor), NKG2D, NKp46, CD2, CD28 and CD25.

The entity that is targeted by the immune cell binding region of thefurther moiety is preferably one which, when bound, leads to activationof the corresponding immune cell (eg T cell). Activation of an immunecell (eg T cell) can be determined by contacting isolated peripheralmononuclear blood cells with the further moiety comprising the immunecell binding region and using standard assays for cell proliferationknown in the art.

Suitable assays for determining the extent of an immune response includeELISpot, intracellular cytokine staining, HLA-peptide tetramer staining,proliferation assay, activation assays (eg CD69), CD107 mobilisationassays or metabolic assays (eg MTT). Also suitable are assays to detectactivation-induced secreted cytokines, for example using ELISA ormultiplexed bead technologies.

For instance, in the preferred embodiment when the immune cell bindingregion of the further moiety is a T cell binding region, it is preferredif the T cell binding region is capable of binding to the CD3 antigenand/or TCR on T cells which are known to activate T cells. CD3 ispresent on all T cells and consists of subunits designated γ, δ, ε, ζand η. The cytoplasmic tail of CD3 is sufficient to transduce thesignals necessary for T cell activation in the absence of the othercomponents of the TCR receptor complex. Normally, activation of T cellcytotoxicity depends first on binding of the TCR with a majorhistocompatibility complex (MHC) protein, itself bound to a foreignantigen, located on a separate cell. Only when this initial TCR-MHCbinding has taken place can the CD3-dependent signally cascaderesponsible for T cell clonal expansion and, ultimately, T cellcytotoxicity ensue. However, when the further moiety of the molecule ofthe present invention binds to CD3 and/or the TCR, activation ofcytotoxic T cells in the absence of independent TCR-MHC can take placeby virtue of the crosslinking of the CD3 and/or TCR molecules mimickingan immune synapse formation. This means that T cells may becytotoxically activated in a clonally independent fashion, ie in amanner which is independent of the specific TCR clone carried by the Tcell. This allows for activation of the entire T cell compartment ratherthan only specific T cells of a certain clonal identity.

Conveniently, therefore, the immune cell binding region (eg T cellbinding region) is one that binds to the immune cell in a way thatmimicks the natural antigen binding to that immune cell which is knownto activate the immune cell, eg to exert its effect on the viability ofthe target cell such as cytotoxicity.

The further moiety may be a polypeptide or a peptide. Preferably, thefurther moiety that contains the immune cell binding region is anantibody that specifically binds to an entity expressed by an immunecell (eg T cell). In a particularly preferred embodiment, the furthermoiety comprises one or more antibody variable domains (eg VH and VLdomains) that specifically bind to an immune cell antigen. The furthermoiety may comprise aVH domain and a VL domain that, when paired,specifically bind to an immune cell antigen, or it may comprise a singleVH domain or a single VL domain that is capable of binding to an immunecell antigen. Thus, the further moiety may be a single chain antibodyconstruct such as a scFv antibody. It is especially preferred if thefurther moiety is an antibody (e.g. one that comprises at least oneantibody variable domain such as a scFv antibody) that specificallybinds to the CD3 antigen on T cells. A specific example is muromonab,but it will be appreciated that any anti-CD3 antibody may also be used.As will become clearer below and from the Figures, the further moietymay comprise two or more separate parts, such as polypeptide domainsthat are not necessarily encoded by contiguous polynucleotide sequences.

By the term ‘masked’, we include the meaning that the immune cellbinding functionality of the immune cell binding region in the furthermoiety is substantially reduced or preferably inhibited completely. Themasking of the immune cell binding region may be reversed by selectiveunmasking in the vicinity of the unwanted cells. Thus, in the maskedstate, the immune cell binding region is preferably unable to bind to animmune cell, while in the unmasked state the immune cell binding regionis able to bind to an immune cell.

As described further below and exemplified in the figures, in aparticularly preferred embodiment, the masking of the immune cellbinding region is controlled by selective cleavage of one or morecleavage sites in the molecule. Thus, the molecule may contain one ormore cleavage sites that are selectively cleavable when the molecule isin the vicinity of the unwanted cells. Suitable cleavage sites andagents that may cleave them are described in more detail below.

When the immune cell binding region is masked, the further moietytypically binds to an immune cell with an affinity at least 5 times lessthan when the immune cell binding region is unmasked, and more typicallyat least 10, 50 or 100 times less than when the immune cell bindingregion is unmasked. Most preferably, there is no detectable bindingbetween the further moiety and an immune cell when the immune cellbinding region is masked. Methods for assessing binding of moieties tocells are common in the art and include, for example, radiolabelledassays, fluorescently labelled techniques and flow cytometry.Conveniently, the further moiety is labelled and added to an immune cellunder conditions conducive to binding in both the unmasked and maskedstates, and the extent of binding between the immune cell and furthermoiety in the masked and unmasked states compared. Biophysicaltechniques such as fluorescence correlation spectroscopy, fluorescenceresonance energy transfer and analytical ultracentrifugation may also beused.

Preferably, binding of the immune cell binding region to an immune cellactivates the immune cell. Thus, when the immune cell binding region ismasked, the further moiety typically activates an immune cell at least 5times less than when the immune cell binding region is unmasked, andmore typically at least 10, 50 or 100 times less than when the immunecell binding region is unmasked. Most preferably, there is no detectableactivation of immune cells when the immune cell binding region ismasked. Methods for assessing activation of immune cells are standard inthe art and include cell proliferation and cytokine secretion assays asdescribed above.

In one embodiment, the immune cell binding region is masked by virtue ofthe further moiety being locked in a particular conformation in whichthe immune cell binding region is not accessible to an immune cell. Theimmune cell binding region may then be unmasked, for example, byselective cleavage of one or more cleavage sites in the molecule, whenin the vicinity of the unwanted cells, which induces a conformationalchange in the further moiety that unmasks the immune cell bindingregion.

An example of this embodiment is shown in FIG. 1, where each of thetargeting moiety and further moiety are respective scFv antibodiesexpressed on a single polypeptide chain. In FIG. 1, the targeting moiety(1) corresponds to one scFv unit that comprises two antibody variabledomains (depicted as ovals in the figures): a first heavy chain variabledomain (VH) and a first light chain variable domain (VL), which domainscan pair together so as to form a first functional epitope binding sitespecific for an antigen expressed on an unwanted cell. The targetingmoiety is attached to a further moiety (3) which corresponds to anotherscFv unit that comprises two antibody variable domains (depicted asovals in the figures): a second VH domain and a second VL domain whichcan pair together so as to form a second functional epitope binding sitespecific for an antigen expressed on an immune cell (e.g. CD3 on a Tcell). Thus, in FIG. 1, the immune cell binding region (4) of thefurther moiety corresponds to the second functional epitope binding sitethat is formed when the second VH domain pairs with the second VLdomain. The second VH domain and second VL domain of the further moiety(3) in FIG. 1 are covalently joined by a polypeptide linker that is ofan insufficient length to allow pairing between the two domains, whichhas the effect of locking the further moiety (3) in a particularconformation in which the immune cell binding region (4) is masked.However, when the molecule is in the vicinity of the unwanted cells (2),cleavage of a protease cleavage site (5) within the polypeptide linkerjoining the second VH domain and second VL domain, breaks the covalentlinkage between the second VH domain and second VL domain so that thedomains can pair and form the second functional epitope binding site. Inother words, cleavage of the protease cleavage site has led to aconformation change in the further moiety (3) which unmasks the immunecell binding region (4).

Accordingly and as exemplified in FIG. 1, in one embodiment, the furthermoiety is a scFv antibody (3) in which the linker that joins the VH andVL domains is of insufficient length to allow pairing of the VH and VLdomains such that the scFv antibody (3) cannot bind to the immune cell,and wherein selective cleavage of one or more cleavage sites (5) in saidlinker, when in the vicinity of the unwanted cells (2), induces pairingof the VH and VL domains such that the scFv antibody (3) can bind to theimmune cell. Preferably, the immune cell binding region is a T cellbinding region such as one that binds to the CD3 antigen on a T cell.

Preferably, the linker in this and other embodiments described herein isa peptide linker. However other linkers including polymers, nucleotides,nucleic acids, polysaccharide chains, lipid organic species (egpolyethylene glycol) may also be used.

Importantly, the linker that joins the VH and VL domains of the furthermoiety must be of insufficient length to allow pairing between the VHand VL domains.

By ‘pairing between VH and VL domains’ we include the meaning ofcorrectly juxtaposing the matched VH and VL domains in a conformationthat is identical to or approximates to the native paired state in acorresponding whole IgG parent antibody or scFv antibody such that thepaired domains bind antigen with similar affinity to the correspondingwhole IgG parent antibody or scFv antibody. Pairing of VH and VL domainsmay be assessed by any binding assay that monitors antibody/antigenbinding, known in the art. Suitable techniques include ELISA, SPR, flowcytometry, FRET and structural studies. Competitive assays may also beemployed where an excess of one or other of the VH or VL domains isused. If the excess causes binding to increase, this would imply thatdomains are unpaired. Additionally, functional immune cell assays may beused to assess pairing of VH and VL domains of the further moiety byinvestigating their ability to bind to, and activate, the correspondingimmune cell. For example, if the VH and VL domains of the further moietywere specific for CD3 antigen on T cells, it may be appropriate tomonitor correct pairing by performing functional T cell assays.

Generally, peptide linkers that join the VH domain and VL domain of ascFv antibody which are 14 amino acids or less are of insufficientlength to allow pairing between the VH and VL domains. Thus, when thelinker is a peptide, the peptide linker is typically 14 amino acids orless, such as 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid inlength.

Preferably, the targeting moiety of the embodiment in FIG. 1 comprises aVH domain and a VL domain which domains can pair together so as to forma functional epitope binding site specific for an antigen expressed onan unwanted cell. In this case, it is understood that the targetingmoiety may be joined to the further moiety such that the molecule can beexpressed as a single polypeptide chain.

In an alternative embodiment, the immune cell binding region is maskedby virtue of the molecule comprising one or more masking moieties thatprevent access of the immune cell binding region to the T cell. Thus,the molecule may comprise at least 1 or 2 or 3 or 4 or more maskingmoieties.

The one or more masking moieties may be any chemical moiety including apolypeptide or a peptide or an antibody or a small molecule. It isappreciated that any moiety that sterically blocks the immune cellbinding region of the further moiety may be used as a masking moiety.Examples of suitable polypeptides include albumin and haemoglobulin.Such moieties can be readily identified by the skilled person. Forexample, antibodies against an antibody known to activate an immune cell(e.g a T cell activating scFv fragment) may be selected by phage displaytechnology or by immunising a mouse with the appropriate antibody.Similarly, small molecule libraries may be screened to identifyinhibitors of a known activator of an immune cell, for example in immunecell activation assays. Other techniques that may be used includemolecular computer modelling and ribosomal display methods.

In one embodiment, and as exemplified in FIGS. 1, 2 and 4, the one ormore masking moieties are immunoglobulin domains. The immunoglobulindomains may be either heavy chain or light chain domains derived from anantibody, and equally they may be either variable or constant domainsderived from an antibody. Thus, the one or more masking moieties may beany one or more of a heavy chain variable domain (VH), a light chainvariable domain (VL), a heavy chain constant domain (CH) and a lightchain constant domain (CL). For example, the molecule may comprise twomasking moieties which are a VL domain and a VH domain, or which are aCL domain and a CH domain. When the one or more masking moieties are aVL domain and a VH domain, the two domains may correspond to a scFvconstruct. It will be appreciated that when the one or more maskingmoieties are immunoglobulin domains, they may have an idiotype specificfor the immune cell binding region of the targeting moiety. Preferably,however, the immunoglobulin domains used as masking moieties do notspecifically bind any other protein in the body, and so are ones thathave low toxicity.

In another embodiment, and as exemplified in FIG. 3, the masking moietyis an immune cell surface antigen which the immune cell binding regionof the further moiety is capable of binding to. For example, if theimmune cell binding region of the further moiety binds to CD3 antigen ona T cell, the masking moiety may be the CD3 antigen (or one of thesubunits thereof). In this scenario, the masking moiety competes withthe T cell for binding to the immune cell binding region of the furthermoiety. It will be appreciated that the masking moiety need not be theentire immune cell surface antigen but may comprise only a portion ofthe immune cell surface antigen provided that the portion is capable ofbinding to the immune cell binding region. Suitable portions include theexternal parts of the immune cell surface antigens or portions thereofthat can bind to the immune cell binding region. Thus, an external partof one of the subunits of the CD3 antigen (eg CD ε) may be used. Variousmethods may be used to determine binding between an immune cell surfaceantigen and an immune cell binding region, including, for example,enzyme linked immunosorbent assays (ELISA), surface plasmon resonanceassays, chip-based assays, immunocytofluorescence, yeast two-hybridtechnology and phage display which are common practice in the art andare described, for example, in Plant et al (1995) Analyt Biochem,226(2), 342-348. and Sambrook et al (2001) Molecular Cloning ALaboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. Such portions of immune cell surface antigensare included in the definition immune cell surface antigen. Accordingly,the masking moiety may comprise an antigenic portion of the immune cellsurface antigen (eg CD3 antigen). Generally, such portions comprise astretch of amino acid residues of at least 8 amino acids, generally atleast 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and moregenerally at least 25, 50, 75 or 100 amino acids.

Typically, the one or more masking moieties are covalently attached tothe molecule by one or more linkers. Such linkers may contain the one ormore cleavage sites which are selectively cleavable when the molecule isin the vicinity of the unwanted cells, as described further below.Alternatively, the one or more masking moieties may be non-covalentlyattached to the molecule. In this case, the one or more cleavage sitesdescribed below may be located at one or more positions in the furthermoiety, such that upon cleavage, the part of the further moiety that isnon-covalently bound to a masking moiety is released from the molecule,and the part of the further moiety that contains the immune cell bindingregion is retained in the molecule. As such, cleavage of the one or morecleavage sites has caused the immune cell binding region to be unmasked.

The one or more masking moieties may operate by simply blocking theimmune cell binding region, so that the region is not accessible to animmune cell. In this instance there is no conformational change in thefurther moiety upon unmasking, and removal of the one or more maskingmoieties simply exposes the underlying T cell binding region so that itmay bind an immune cell. It will be appreciated that the masking moietymay be any suitable chemical moiety that sterically blocks the immunecell region, as discussed above. Examples of this embodiment are shownin FIGS. 2 and 3, where the masking moiety is either an immunoglobulindomain or an immune cell surface antigen respectively.

In FIG. 2, as in FIG. 1, each of the targeting moiety and further moietycomprise respective pairs of VH and VL domains. Although FIG. 2 depictsa molecule containing two polypeptide chains, it will be appreciatedthat each of the targeting moiety and further moiety may also berespective scFv antibodies that are expressed on a single polypeptidechain. In the figure, the targeting moiety (1) comprises a first VHdomain and a first VL domain which domains can pair together so as toform a first functional epitope binding site specific for an antigenexpressed on an unwanted cell. The targeting moiety (1) is attached to afurther moiety (3) that comprises a second VH domain and a second VLdomain which can pair together so as to form a second functional epitopebinding site specific for an antigen expressed on an immune cell (e.g.CD3), i.e. the immune cell binding region (4). However, the immune cellbinding region (4) is masked by virtue of the second VH domain andsecond VL domain of the further moiety being joined to, respectively, afirst masking moiety (6) comprising a CH domain and a second maskingmoiety (6) comprising a CL domain, which act to block the immune cellbinding region. When the molecule is in the vicinity of the unwantedcells, cleavage of the protease cleavage sites (5) within the respectivepolypeptide linkers joining the second VH domain to the CH domain of thefirst masking moiety, and the second VL domain to the CL domain of thesecond masking moiety, acts to release the CH domain and CL domainmasking moieties (6), such that the second functional epitope bindingsite becomes accessible to an immune cell. In other words, cleavage ofthe protease cleavage sites has unmasked the immune cell binding region.

Accordingly, and as exemplified in FIG. 2, in one embodiment, themolecule of the invention comprises:

-   -   a further moiety (3) which comprises a VH domain and a VL domain        that are capable of specifically binding to an immune cell,    -   a first linker which joins the VH domain of the further moiety        (3) to a CH domain of a first masking moiety (6), and    -   a second linker which joins the VL domain of the further moiety        (3) to a CL domain of a second masking moiety (6),    -   such that the CH and CL domains of the first and second masking        moieties (6) mask the binding regions of the VH and VL domains        (4) so as to prevent binding of the further moiety to an immune        cell, and    -   wherein selective cleavage of one or more cleavage sites in said        linkers (5), when in the vicinity of the unwanted cells (2),        releases the first and second masking moieties (6) from the        further moiety (3) so as to allow binding of the further moiety        (3) to an immune cell. Preferably, the immune cell binding        region is a T cell binding region such as one that binds to the        CD3 antigen on a T cell. Preferably, the targeting moiety        comprises a VH domain and a VL domain which domains can pair        together so as to form a functional epitope binding site        specific for an antigen expressed on an unwanted cell. The        targeting moiety may or may not be expressed as a single        polypeptide with the further moiety. It is appreciated in this        embodiment that the first and second masking moieties may be any        chemical moieties capable of preventing binding of the immune        cell binding region to the immune cell, for example by steric        blocking. For the avoidance of doubt therefore, although FIG. 2        depicts the masking moieties as CH and CL domains, it is        understood that any one or more suitable masking moieties may be        used, including any combination of immunoglobulin domains.

In FIG. 3, each of the targeting moiety and further moiety arerespective scFv antibodies expressed as a single polypeptide chain.However, it will be appreciated that the respective VH and VL domains ofthe targeting moiety and further moiety of the molecule shown in FIG. 3may equally be present on two polypeptide chains that pair together (egscFv-like molecules or diabodies). In the figure, the targeting moiety(1) corresponds to one scFv unit that comprises a first VH domain and afirst VL domain which domains can pair together so as to form a firstfunctional epitope binding site specific for an antigen expressed on anunwanted cell. The targeting moiety is attached to a further moiety (3)that corresponds to another scFv unit which comprises a second VH domainand a second VL domain which can pair together so as to form a secondfunctional epitope binding site specific for an antigen expressed on animmune cell (e.g. CD3), i.e. the immune cell binding region (4).However, the immune cell binding region is masked by virtue of a linkerjoining one of the second VH domain or second VL domain of the furthermoiety (3) to an immune cell surface antigen, i.e. the masking moiety(6), such that the immune cell surface antigen binds to and blocks theimmune cell binding region (4). When the molecule is in the vicinity ofthe unwanted cells (2), cleavage of the cleavage site (5) within thelinker allows the immune cell surface antigen (6) to leave the immunecell binding region, such that the second functional epitope bindingsite (4) becomes accessible to an immune cell. In other words, cleavageof the cleavage site (5) has unmasked the immune cell binding region(4). This embodiment involves competition between an immune cell and theimmune cell surface antigen (6) within the molecule for binding to theimmune cell binding region (4) of the further moiety (3). When not inthe vicinity of unwanted cells (2), the immune cell surface antigen (6)is joined to the molecule by means of a linker which increases the localconcentration of the immune cell surface antigen (6) and so favoursbinding of the immune cell surface antigen (6), rather than an immunecell, to the immune cell binding region (4). When the molecule is in thevicinity of unwanted cells (2), the linker joining the immune cellsurface antigen (6) to the molecule is cleaved and so the immune cellsurface antigen (6) is free to leave the immune cell binding region (4)such that an immune cell can now bind to it.

Accordingly, the molecule of the invention may comprise:

-   -   a further moiety (3) which comprises a VH domain and a VL domain        that are capable of specifically binding to an immune cell, and    -   a linker which joins the further moiety (3) to an immune cell        surface antigen (6),    -   such that the immune cell surface antigen (6) binds to and masks        the immune cell binding region (4) of the VH and VL domains so        as to prevent binding of the further moiety (3) to an immune        cell,    -   wherein selective cleavage of one or more cleavage sites (5) in        the linker, when in the vicinity of the unwanted cells (2),        releases the immune cell surface antigen (6) from the immune        cell binding region (4) of the VH and VL domains so as to allow        binding of the further moiety (3) to an immune cell. Preferably,        the immune cell binding region (4) is a T cell binding region        such as one which specifically binds to the CD3 antigen of a T        cell, and the immune cell surface antigen is a T cell surface        antigen such as CD3 antigen or part thereof. Preferably, and as        exemplified in FIG. 3, the targeting moiety comprises a VH        domain and a VL domain which domains can pair together so as to        form a functional epitope binding site specific for an antigen        expressed on an unwanted cell. In this way, the targeting moiety        and further moiety may correspond to two separate scFv units        that can be expressed as a single polypeptide chain.

In an alternative embodiment, the one or more masking moieties operateby promoting a conformation of the further moiety such that the immunecell binding region is not accessible to an immune cell. In this case,unmasking does involve a conformational change in the further moietysuch that the immune cell binding region now becomes accessible forbinding to an immune cell. For example, when the further moietycomprises a pair of VH and VL domains that specifically bind to anantigen on an immune cell (e.g. CD3 antigen), it is appreciated that theone or more masking moieties may act to prevent pairing of the componentVH and VL domains of the further moiety such that the VH and VL domainsare not in the desired three dimensional conformation to bind to theimmune cell antigen. Various methods may be used to prevent pairing ofthe VH and VL domains of a further moiety. One such method isexemplified in FIG. 4 which makes use of dummy variable domains thatpair with the VH and VL domains of the further moiety such that the VHand VL domains of the further moiety cannot pair with each other.However, any suitable masking moiety that prevents pairing of the VH andVL domains of the further moiety, for example by binding to VH-VLinterface protein surface, may be used. In all cases, the prevention ofpairing of the VH and VL domains is lifted by cleavage of one or morecleavage sites within the molecule when the molecule is in the vicinityof the wanted cells.

In FIG. 4, the targeting moiety (1) corresponds to a scFv unit thatcomprises a first VH domain and a first VL domain which domains can pairtogether so as to form a first functional epitope binding site specificfor an antigen expressed on an unwanted cell. A first linker joins thefirst VL domain of the targeting moiety (1) to a second VH domain of afurther moiety (3), a second linker joins the first VH domain of thetargeting moiety (1) to a first masking moiety comprising a VH domain(6), a third linker joins the second VH domain of the further moiety (3)to a second masking moiety comprising a VL domain (6), and a fourthlinker joins the VH domain of the first masking moiety to a second VLdomain of the further moiety (3); wherein the second VH and second VLdomains of the further moiety, when paired, form a second functionalepitope binding site specific for an antigen expressed on an immune cell(e.g. CD3 antigen on a T cell), i.e. the immune cell binding region (4);and wherein the third and fourth linkers are of a sufficient length sothat the second VH domain of the further moiety (3) is paired with theVL domain of the second masking moiety (6), and the second VL domain ofthe further moiety (3) is paired with the VH domain of the first maskingmoiety (6). Thus, the first and second masking moieties in FIG. 4prevent the second VH and second VL domain of the further moiety frompairing and so mask the immune cell binding region (4). In this way, thevariable domains of the masking moieties act as ‘dummy’ VH and VLdomains which pair with the second VH and VL domains of the furthermoiety, and the third and fourth linkers stabilise this configuration bypreventing domain exchange. However, when the molecule is in thevicinity of the unwanted cells (2), cleavage of one or more cleavagesites (5) present in the third and fourth linkers release the second VHdomain and second VL domain from pairing with the VL domain of thesecond masking moiety (6) and the VH domain of the first masking moiety(6), respectively, so that the second VH domain and second VL domain ofthe further moiety can pair to form the immune cell binding region (4).In other words, cleavage of the cleavage sites (5) present in the thirdand fourth linkers has allowed conformational rearrangement of themolecule wherein the second VH domain and second VL domain are correctlypaired so that the immune cell binding ability is recovered. The immunecell binding region (4) has been unmasked.

Accordingly, in one embodiment, the molecule of the invention comprises:

-   -   a further moiety (3) which comprises separately a first VH and a        first VL domain which when paired are capable of specifically        binding to an immune cell,    -   a linker which joins the first VH domain to a second VL domain        of a first masking moiety (6), and    -   a linker which joins the first VL domain to a second VH domain        of a second masking moiety (6),    -   such that the first VH and first VL domains of the further        moiety are not paired and the further moiety cannot bind to the        immune cell, and    -   wherein selective cleavage of one or more cleavage sites (5) in        said linkers, when in the vicinity of the unwanted cells (2),        allows pairing of the first VH and first VL domains such that        the further moiety can bind to the immune cell. Preferably, the        immune cell binding region (4) is a T cell binding region such        as one that specifically binds to the CD3 antigen on a T cell.        Given that the masking moieties mask the immune cell binding        region, it will of course be appreciated in this embodiment,        that the second VH domain and second VL domain of the masking        moieties are not ones which are capable of respectively pairing        with the first VL domain and first VH domain of the further        moiety so as to form a functional epitope binding site specific        for an antigen expressed on an immune cell (e.g. an antigen        expressed on a T cell such as CD3 antigen). In any event, it        will further be appreciated that any suitable masking moiety        that prevents pairing of the VH and VL domains of the further        moiety may be substituted for the VH and VL masking moieties        shown in FIG. 4. Preferably, and as exemplified in FIG. 4, the        targeting moiety comprises a VH domain and a VL domain which        domains can pair together so as to form a functional epitope        binding site specific for an antigen expressed on an unwanted        cell. In this case, it is understood that the targeting moiety        may be joined to the further moiety and the one or more masking        moieties such that the molecule can be expressed as a single        polypeptide chain. The targeting moiety may be in the middle of        the polypeptide chain flanked by the further moiety and/or one        or more masking moieties. For example, the targeting moiety may        be a scFv unit, one domain of which is joined to a first VL        domain of the further moiety and the other domain of which is        joined to a second VL domain of a first masking moiety, or the        targeting moiety may be a scFv unit, one domain of which is        joined to the first VH domain of the further moiety and the        other domain of which is joined to second VH domain of the        second masking moiety. Alternatively, the targeting moiety may        be at either end of the polypeptide chain.

Preferably, and as exemplified in FIG. 4, the linker which joins thefirst VH domain to a second VL domain of a first masking moiety, is ofsufficient length to allow the first VH domain to be paired with thesecond VL domain, and the linker which joins the first VL domain to asecond VH domain of a second masking moiety is of sufficient length toallow the first VL domain to be paired with the second VH domain.Typically, the linkers are peptides and so must have a sufficient numberof amino acids to allow pairing between the VH and VL domains.Generally, peptide linkers that join a VH domain and a VL domain of ascFv antibody which are 15 amino acids or more are of sufficient lengthto allow pairing between the VH and VL domains. Thus, when said linkersare peptides, they are typically 15 amino acids or more in length, suchas at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids in length. Pairingbetween VH and VL domains can be assessed by routine methods known inthe art, including those described above.

Selective Unmasking of Immune Cell Binding Region

By ‘selective unmasking’ we include the meaning that the immune cellbinding region is unmasked by the presence of an agent that selectivelyresides in the in the vicinity of the unwanted cells, which agent actsto unmask the immune cell binding region. Preferably, the agent thatunmasks the immune cell binding region resides in the vicinity of theunwanted cells at least five or ten times higher than the concentrationof the agent outside the vicinity of the unwanted cells, and morepreferably at least 100 or 500 or 1000 times higher. Most preferably,the agent that unmasks the immune cell binding region binding region isfound only in the vicinity of the unwanted cells.

In a preferred embodiment, the immune cell binding region is unmasked byselective cleavage of one or more cleavage sites in the molecule when inthe vicinity of the unwanted cells. In this case, the agent is one thatcleaves the one or more cleavage sites. In other words, the immune cellbinding region may be unmasked by means of one or more cleavage siteswithin the molecule being cleaved selectively in the vicinity of theunwanted cells.

Thus, the invention includes a molecule comprising (i) a targetingmoiety capable of directly or indirectly targeting to unwanted cells,and (ii) a further moiety that has a masked immune cell binding regionso as to prevent binding of the further moiety to an immune cell,wherein selective cleavage of one or more cleavage sites in the moleculewhen in the vicinity of the unwanted cells unmasks the immune cellbinding region so as to allow binding of the further moiety to an immunecell. Preferably, the immune cell binding region is a T cell bindingregion including one that binds to the CD3 antigen and/or TCR on a Tcell. Accordingly, it is appreciated that the invention provides amolecule comprising (i) a targeting moiety capable of directly orindirectly targeting to unwanted cells, and (ii) a further moiety thathas a masked T cell binding region so as to prevent binding of thefurther moiety to a T cell, wherein selective cleavage of one or morecleavage sites in the molecule when in the vicinity of the unwantedcells unmasks the T cell binding region so as to allow binding of thefurther moiety to a T cell.

By “cleavage site that is cleavable selectively in the vicinity of theunwanted cells” we include the meaning of a site that can only becleaved by an agent which resides selectively in the vicinity of theunwanted cells, so as to unmask the immune cell binding region.Preferably, the agent that cleaves the one or more cleavage sitesresides in the vicinity of the unwanted cells at a concentration atleast five times or ten times higher than the concentration of the agentoutside the vicinity of the unwanted cells, and more preferably at aconcentration at least 100 or 500 or 1000 times higher. Most preferably,the agent that cleaves the one or more cleavage sites is found only inthe vicinity of the unwanted cells. For example, when the unwanted cellsare particular tumour cells (e.g. breast tumour cells), the one or morecleavage sites may be ones that are cleaved by an agent which residesselectively in the particular tumour (e.g. breast tumour) but whichagent does not reside outside the vicinity of the particular tumour(e.g. breast tumour).

By in the vicinity of cells′, we include the meaning of either at ornear to the surface of the cells, or both, or in the environment thatimmediately surrounds the cells e.g. blood, lymph, and other bodyfluids.

The one or more cleavage sites are selectively cleaved in the vicinityof the unwanted cells so that the immune cell binding region of thefurther moiety is preferentially unmasked in the vicinity of theunwanted cells so as to recruit immune cells preferentially to unwantedcells rather than wanted cells. Thus, it is preferred that the one ormore cleavage sites are ones that are selectively cleaved such that theimmune cell binding region is unmasked in the vicinity of the unwantedcells at least five times or ten times more than the extent to which itis unmasked in the vicinity of wanted cells, and more preferably atleast 100 or 500 or 1000 times more. Most preferably, the immune cellbinding region is not unmasked in the vicinity of wanted cells, andtherefore immune cells are not recruited to wanted cells.

For a given unwanted cell, the skilled person will be able to identifyappropriate one or more cleavage sites that are selectively cleavable inthe vicinity of the unwanted cell, using established methods in the art.For example, which proteases cleave which peptides can be assessed byconsulting peptide libraries and studying an MS analysis of thefragmentation profile following cleavage. Also, published literature ofprotease cleavage motifs and peptide cleavage data can be searched asdescribed further below. Gene expression and proteomic data may also beanalysed to identify which proteases are expressed by particularunwanted cells.

By virtue of the one or more cleavage sites being selectively cleavablein the vicinity of the unwanted cells, the immune cell binding region isselectively unmasked in the vicinity of the unwanted cells.

The one or more cleavage sites may be located between separate parts ofthe further moiety (e.g. separate immunoglobulin domains), such thatupon cleavage of the cleavage sites a conformational change is inducedwhereby the separate parts of the further moiety are rearranged so as tounmask the immune cell binding region (see FIG. 1). The one or morecleavage sites may be located between the one or more masking moietiesand either the further moiety and/or targeting moiety. Thus, the one ormore cleavage sites may be located between the one or more maskingmoieties and the further moiety (see FIGS. 2 and 3), or they may belocated between the one or more masking moieties and the targetingmoiety (see FIG. 4), or they may be located between the one or moremasking moieties and each of the targeting moiety and further moiety(see FIG. 4). When any of the further moiety, targeting moiety andmasking moiety comprise one or more immunoglobulin domains, it isappreciated that the one or more cleavage sites should be locatedbetween the immunoglobulin domains to allow movement of wholeimmunoglobulin domains rather than movement of only parts of thedomains, upon unmasking of the immune cell binding region.

The cleavage site may be one that is cleavable by an enzyme such as anyof a protease, a nuclease, a lipase, a lyase, a phosphatase or acarbohydrase, which may or may not be membrane bound. Accordingly, itwill be appreciated that the cleavage site may be one that is cleavableby an enzyme that is bound to the membrane of, or secreted by, theunwanted cell.

Generally, the cleavage site is a protease cleavage site. Thus, when theunwanted cells are tumour cells, the one or more cleavage sites may becleavable selectively by proteases that reside in the vicinity of thetumour cells. In other words, the protease cleavage site may be one thatis cleavable by a tumour associated protease. It is well known thatduring tumour development, tumours aberrantly express proteases whichallow them to invade local tissues and eventually metastasise.

The protease may include any of a cysteine protease (including theCathepsin family B, L, S etc), an aspartyl protease (including CathepsinD and E) and a serine protease (including Cathepsin A and G, Thrombin,Plasmin, Urokinase, Tissue Plasminogen Activator). The protease may be ametalloproteinase (MMP1-28) including both membrane bound (MMP14-17 andMMP24-25) and secreted forms (MMP1-13 and MMP18-23 and MMP26-28). Theprotease may belong to the A Disintegrin and Metalloproteinase (ADAM)and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs(ADAMTS) families of proteases. Other examples include CD10 (CALLA) andprostate specific antigen (PSA). It is appreciated that the proteasesmay or may not be membrane bound.

Protease cleavage sites are well known in the scientific literature, andlinker sequences comprising such cleavage sites can be readilyconstructed using established genetic engineering techniques, or bysynthetic synthesis techniques known in the art.

Protease cleavage sites may be ones that are cleavable by any of theproteases listed in Table 4 below, which indicates the expression ofselected proteases in various tumour types. Candidate substrates for theproteases are provided. Thus, in order to treat a particular tumourtype, the skilled person will typically select one or more proteasecleavage sites that are selectively cleaved by a protease known to behighly expressed in that tumour type, as seen from the table. Forexample, to treat breast cancer, it is preferred to use a proteasecleavage site cleavable by any of uPA, tPA, matriptase, matriptase 2,Cathepsin K, Cathepsin 0, MMP1, MMP2, MMP3, MMP11, MMP12, MMP17, ADAM9,ADAM12, ADAM15, ADAM17, ADAM28 or ADAMTS15, and so on. It will beappreciated that the one or more protease cleavage sites selected by theskilled person include ones that are cleaved by different proteases,which may help to improve the specificity of the molecule of theinvention further.

Similarly, Table 5 lists tumour sites in which ADAM proteaseoverexpression has been reported, and so in an embodiment, the one ormore cleavage sites are selectively cleavable by one of the ADAMproteases listed in Table 5. Accordingly, the molecule may be used toprevent or treat the corresponding tumour type.

The one or more cleavage sites may be selectively cleavable by any ofthe following human proteases (MEROPS peptidase database number providedin parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. &Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res.36 Database issue, D320-325): pepsin A (MER000885), gastricsin(MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D(MER000911), cathepsin E (MER000944), memapsin-1 (MER005534), napsin A(MER004981), Mername-AA034 peptidase (MER014038), pepsin A4 (MER037290),pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens)-typeputative peptidase (MER107386), napsin B pseudogene (MER004982), CYMPg.p. (Homo sapiens) (MER002929), subfamily A1A unassigned peptidases(MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbitendogenous retrovirus endopeptidase (MER043650), S71-related humanendogenous retropepsin (MER001812), RTVL-H-type putative peptidase(MER047117), RTVL-H-type putative peptidase (MER047133), RTVL-H-typeputative peptidase (MER047160), RTVL-H-type putative peptidase(MER047206), RTVL-H-type putative peptidase (MER047253), RTVL-H-typeputative peptidase (MER047260), RTVL-H-type putative peptidase(MER047291), RTVL-H-type putative peptidase (MER047418), RTVL-H-typeputative peptidase (MER047440), RTVL-H-type putative peptidase(MER047479), RTVL-H-type putative peptidase (MER047559), RTVL-H-typeputative peptidase (MER047583), RTVL-H-type putative peptidase(MER015446), human endogenous retrovirus retropepsin homologue 1(MER015479), human endogenous retrovirus retropepsin homologue 2(MER015481), endogenous retrovirus retropepsin pseudogene 1 (Homosapiens chromosome 14) (MER029977), endogenous retrovirus retropepsinpseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenousretrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17)(MER002660), endogenous retrovirus retropepsin pseudogene 3 (Homosapiens chromosome 17) (MER030286), endogenous retrovirus retropepsinpseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenousretrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12)(MER029664), endogenous retrovirus retropepsin pseudogene 6 (Homosapiens chromosome 7) (MER002094), endogenous retrovirus retropepsinpseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenousretrovirus retropepsin pseudogene 8 (Homo sapiens chromosome Y)(MER030291), endogenous retrovirus retropepsin pseudogene 9 (Homosapiens chromosome 19) (MER029680), endogenous retrovirus retropepsinpseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenousretrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17)(MER004378), endogenous retrovirus retropepsin pseudogene 12 (Homosapiens chromosome 11) (MER003344), endogenous retrovirus retropepsinpseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779),endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome2) (MER029778), endogenous retrovirus retropepsin pseudogene 15 (Homosapiens chromosome 4) (MER047158), endogenous retrovirus retropepsinpseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenousretrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4)(MER003182), endogenous retrovirus retropepsin pseudogene 16(MER047165), endogenous retrovirus retropepsin pseudogene 16(MER047178), endogenous retrovirus retropepsin pseudogene 16(MER047200), endogenous retrovirus retropepsin pseudogene 16(MER047315), endogenous retrovirus retropepsin pseudogene 16(MER047405), endogenous retrovirus retropepsin pseudogene 16(MER030292), endogenous retrovirus retropepsin pseudogene 17 (Homosapiens chromosome 8) (MER005305), endogenous retrovirus retropepsinpseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenousretrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16)(MER001740), endogenous retrovirus retropepsin pseudogene 21 (Homosapiens) (MER047222), endogenous retrovirus retropepsin pseudogene 21(Homo sapiens) (MER047454), endogenous retrovirus retropepsin pseudogene21 (Homo sapiens) (MER047477), endogenous retrovirus retropepsinpseudogene 21 (Homo sapiens) (MER004403), endogenous retrovirusretropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287),subfamily A2A non-peptidase homologues (MER047046), subfamily A2Anon-peptidase homologues (MER047052), subfamily A2A non-peptidasehomologues (MER047076), subfamily A2A non-peptidase homologues(MER047080), subfamily A2A non-peptidase homologues (MER047088),subfamily A2A non-peptidase homologues (MER047089), subfamily A2Anon-peptidase homologues (MER047091), subfamily A2A non-peptidasehomologues (MER047092), subfamily A2A non-peptidase homologues(MER047093), subfamily A2A non-peptidase homologues (MER047094),subfamily A2A non-peptidase homologues (MER047097), subfamily A2Anon-peptidase homologues (MER047099), subfamily A2A non-peptidasehomologues (MER047101), subfamily A2A non-peptidase homologues(MER047102), subfamily A2A non-peptidase homologues (MER047107),subfamily A2A non-peptidase homologues (MER047108), subfamily A2Anon-peptidase homologues (MER047109), subfamily A2A non-peptidasehomologues (MER047110), subfamily A2A non-peptidase homologues(MER047111), subfamily A2A non-peptidase homologues (MER047114),subfamily A2A non-peptidase homologues (MER047118), subfamily A2Anon-peptidase homologues (MER047121), subfamily A2A non-peptidasehomologues (MER047122), subfamily A2A non-peptidase homologues(MER047126), subfamily A2A non-peptidase homologues (MER047129),subfamily A2A non-peptidase homologues (MER047130), subfamily A2Anon-peptidase homologues (MER047134), subfamily A2A non-peptidasehomologues (MER047135), subfamily A2A non-peptidase homologues(MER047137), subfamily A2A non-peptidase homologues (MER047140),subfamily A2A non-peptidase homologues (MER047141), subfamily A2Anon-peptidase homologues (MER047142), subfamily A2A non-peptidasehomologues (MER047148), subfamily A2A non-peptidase homologues(MER047149), subfamily A2A non-peptidase homologues (MER047151),subfamily A2A non-peptidase homologues (MER047154), subfamily A2Anon-peptidase homologues (MER047155), subfamily A2A non-peptidasehomologues (MER047156), subfamily A2A non-peptidase homologues(MER047157), subfamily A2A non-peptidase homologues (MER047159),subfamily A2A non-peptidase homologues (MER047161), subfamily A2Anon-peptidase homologues (MER047163), subfamily A2A non-peptidasehomologues (MER047166), subfamily A2A non-peptidase homologues(MER047171), subfamily A2A non-peptidase homologues (MER047173),subfamily A2A non-peptidase homologues (MER047174), subfamily A2Anon-peptidase homologues (MER047179), subfamily A2A non-peptidasehomologues (MER047183), subfamily A2A non-peptidase homologues(MER047186), subfamily A2A non-peptidase homologues (MER047190),subfamily A2A non-peptidase homologues (MER047191), subfamily A2Anon-peptidase homologues (MER047196), subfamily A2A non-peptidasehomologues (MER047198), subfamily A2A non-peptidase homologues(MER047199), subfamily A2A non-peptidase homologues (MER047201),subfamily A2A non-peptidase homologues (MER047202), subfamily A2Anon-peptidase homologues (MER047203), subfamily A2A non-peptidasehomologues (MER047204), subfamily A2A non-peptidase homologues(MER047205), subfamily A2A non-peptidase homologues (MER047207),subfamily A2A non-peptidase homologues (MER047208), subfamily A2Anon-peptidase homologues (MER047210), subfamily A2A non-peptidasehomologues (MER047211), subfamily A2A non-peptidase homologues(MER047212), subfamily A2A non-peptidase homologues (MER047213),subfamily A2A non-peptidase homologues (MER047215), subfamily A2Anon-peptidase homologues (MER047216), subfamily A2A non-peptidasehomologues (MER047218), subfamily A2A non-peptidase homologues(MER047219), subfamily A2A non-peptidase homologues (MER047221),subfamily A2A non-peptidase homologues (MER047224), subfamily A2Anon-peptidase homologues (MER047225), subfamily A2A non-peptidasehomologues (MER047226), subfamily A2A non-peptidase homologues(MER047227), subfamily A2A non-peptidase homologues (MER047230),subfamily A2A non-peptidase homologues (MER047232), subfamily A2Anon-peptidase homologues (MER047233), subfamily A2A non-peptidasehomologues (MER047234), subfamily A2A non-peptidase homologues(MER047236), subfamily A2A non-peptidase homologues (MER047238),subfamily A2A non-peptidase homologues (MER047239), subfamily A2Anon-peptidase homologues (MER047240), subfamily A2A non-peptidasehomologues (MER047242), subfamily A2A non-peptidase homologues(MER047243), subfamily A2A non-peptidase homologues (MER047249),subfamily A2A non-peptidase homologues (MER047251), subfamily A2Anon-peptidase homologues (MER047252), subfamily A2A non-peptidasehomologues (MER047254), subfamily A2A non-peptidase homologues(MER047255), subfamily A2A non-peptidase homologues (MER047263),subfamily A2A non-peptidase homologues (MER047265), subfamily A2Anon-peptidase homologues (MER047266), subfamily A2A non-peptidasehomologues (MER047267), subfamily A2A non-peptidase homologues(MER047268), subfamily A2A non-peptidase homologues (MER047269),subfamily A2A non-peptidase homologues (MER047272), subfamily A2Anon-peptidase homologues (MER047273), subfamily A2A non-peptidasehomologues (MER047274), subfamily A2A non-peptidase homologues(MER047275), subfamily A2A non-peptidase homologues (MER047276),subfamily A2A non-peptidase homologues (MER047279), subfamily A2Anon-peptidase homologues (MER047280), subfamily A2A non-peptidasehomologues (MER047281), subfamily A2A non-peptidase homologues(MER047282), subfamily A2A non-peptidase homologues (MER047284),subfamily A2A non-peptidase homologues (MER047285), subfamily A2Anon-peptidase homologues (MER047289), subfamily A2A non-peptidasehomologues (MER047290), subfamily A2A non-peptidase homologues(MER047294), subfamily A2A non-peptidase homologues (MER047295),subfamily A2A non-peptidase homologues (MER047298), subfamily A2Anon-peptidase homologues (MER047300), subfamily A2A non-peptidasehomologues (MER047302), subfamily A2A non-peptidase homologues(MER047304), subfamily A2A non-peptidase homologues (MER047305),subfamily A2A non-peptidase homologues (MER047306), subfamily A2Anon-peptidase homologues (MER047307), subfamily A2A non-peptidasehomologues (MER047310), subfamily A2A non-peptidase homologues(MER047311), subfamily A2A non-peptidase homologues (MER047314),subfamily A2A non-peptidase homologues (MER047318), subfamily A2Anon-peptidase homologues (MER047320), subfamily A2A non-peptidasehomologues (MER047321), subfamily A2A non-peptidase homologues(MER047322), subfamily A2A non-peptidase homologues (MER047326),subfamily A2A non-peptidase homologues (MER047327), subfamily A2Anon-peptidase homologues (MER047330), subfamily A2A non-peptidasehomologues (MER047333), subfamily A2A non-peptidase homologues(MER047362), subfamily A2A non-peptidase homologues (MER047366),subfamily A2A non-peptidase homologues (MER047369), subfamily A2Anon-peptidase homologues (MER047370), subfamily A2A non-peptidasehomologues (MER047371), subfamily A2A non-peptidase homologues(MER047375), subfamily A2A non-peptidase homologues (MER047376),subfamily A2A non-peptidase homologues (MER047381), subfamily A2Anon-peptidase homologues (MER047383), subfamily A2A non-peptidasehomologues (MER047384), subfamily A2A non-peptidase homologues(MER047385), subfamily A2A non-peptidase homologues (MER047388),subfamily A2A non-peptidase homologues (MER047389), subfamily A2Anon-peptidase homologues (MER047391), subfamily A2A non-peptidasehomologues (MER047394), subfamily A2A non-peptidase homologues(MER047396), subfamily A2A non-peptidase homologues (MER047400),subfamily A2A non-peptidase homologues (MER047401), subfamily A2Anon-peptidase homologues (MER047403), subfamily A2A non-peptidasehomologues (MER047406), subfamily A2A non-peptidase homologues(MER047407), subfamily A2A non-peptidase homologues (MER047410),subfamily A2A non-peptidase homologues (MER047411), subfamily A2Anon-peptidase homologues (MER047413), subfamily A2A non-peptidasehomologues (MER047414), subfamily A2A non-peptidase homologues(MER047416), subfamily A2A non-peptidase homologues (MER047417),subfamily A2A non-peptidase homologues (MER047420), subfamily A2Anon-peptidase homologues (MER047423), subfamily A2A non-peptidasehomologues (MER047424), subfamily A2A non-peptidase homologues(MER047428), subfamily A2A non-peptidase homologues (MER047429),subfamily A2A non-peptidase homologues (MER047431), subfamily A2Anon-peptidase homologues (MER047434), subfamily A2A non-peptidasehomologues (MER047439), subfamily A2A non-peptidase homologues(MER047442), subfamily A2A non-peptidase homologues (MER047445),subfamily A2A non-peptidase homologues (MER047449), subfamily A2Anon-peptidase homologues (MER047450), subfamily A2A non-peptidasehomologues (MER047452), subfamily A2A non-peptidase homologues(MER047455), subfamily A2A non-peptidase homologues (MER047457),subfamily A2A non-peptidase homologues (MER047458), subfamily A2Anon-peptidase homologues (MER047459), subfamily A2A non-peptidasehomologues (MER047463), subfamily A2A non-peptidase homologues(MER047468), subfamily A2A non-peptidase homologues (MER047469),subfamily A2A non-peptidase homologues (MER047470), subfamily A2Anon-peptidase homologues (MER047476), subfamily A2A non-peptidasehomologues (MER047478), subfamily A2A non-peptidase homologues(MER047483), subfamily A2A non-peptidase homologues (MER047488),subfamily A2A non-peptidase homologues (MER047489), subfamily A2Anon-peptidase homologues (MER047490), subfamily A2A non-peptidasehomologues (MER047493), subfamily A2A non-peptidase homologues(MER047494), subfamily A2A non-peptidase homologues (MER047495),subfamily A2A non-peptidase homologues (MER047496), subfamily A2Anon-peptidase homologues (MER047497), subfamily A2A non-peptidasehomologues (MER047499), subfamily A2A non-peptidase homologues(MER047502), subfamily A2A non-peptidase homologues (MER047504),subfamily A2A non-peptidase homologues (MER047511), subfamily A2Anon-peptidase homologues (MER047513), subfamily A2A non-peptidasehomologues (MER047514), subfamily A2A non-peptidase homologues(MER047515), subfamily A2A non-peptidase homologues (MER047516),subfamily A2A non-peptidase homologues (MER047520), subfamily A2Anon-peptidase homologues (MER047533), subfamily A2A non-peptidasehomologues (MER047537), subfamily A2A non-peptidase homologues(MER047569), subfamily A2A non-peptidase homologues (MER047570),subfamily A2A non-peptidase homologues (MER047584), subfamily A2Anon-peptidase homologues (MER047603), subfamily A2A non-peptidasehomologues (MER047604), subfamily A2A non-peptidase homologues(MER047606), subfamily A2A non-peptidase homologues (MER047609),subfamily A2A non-peptidase homologues (MER047616), subfamily A2Anon-peptidase homologues (MER047619), subfamily A2A non-peptidasehomologues (MER047648), subfamily A2A non-peptidase homologues(MER047649), subfamily A2A non-peptidase homologues (MER047662),subfamily A2A non-peptidase homologues (MER048004), subfamily A2Anon-peptidase homologues (MER048018), subfamily A2A non-peptidasehomologues (MER048019), subfamily A2A non-peptidase homologues(MER048023), subfamily A2A non-peptidase homologues (MER048037),subfamily A2A unassigned peptidases (MER047164), subfamily A2Aunassigned peptidases (MER047231), subfamily A2A unassigned peptidases(MER047386), skin aspartic protease (MER057097), presenilin 1(MER005221), presenilin 2 (MER005223), impas 1 peptidase (MER019701),impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase(MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18)(MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11)(MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsinF (MER004980), cathepsin L (MER000622), cathepsin S (MER000633),cathepsin O (MER001690), cathepsin K (MER000644), cathepsin W(MER003756), cathepsin H (MER000629), cathepsin B (MER000686),dipeptidyl-peptidase I (MER001937), bleomycin hydrolase (animal)(MER002481), tubulointerstitial nephritis antigen (MER016137),tubulointerstitial nephritis antigen-related protein (MER021799),cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsinB-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsinB-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 g.p.(Homo sapiens) (MER005210), CTSLL3 g.p. (Homo sapiens) (MER005209),calpain-1 (MER000770), calpain-2 (MER000964), calpain-3 (MER001446),calpain-9 (MER004042), calpain-8 (MER021474), calpain-15 (MER004745),calpain-5 (MER002939), calpain-11 (MER005844), calpain-12 (MER029889),calpain-10 (MER013510), calpain-13 (MER020139), calpain-14 (MER029744),Mername-AA253 peptidase (MER005537), calpamodulin (MER000718),hypothetical protein flj40251 (MER003201), ubiquitinyl hydrolase-L1(MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinylhydrolase-BAP1 (MER003989), ubiquitinyl hydrolase-UCH37 (MER005539),ubiquitin-specific peptidase 5 (MER002066), ubiquitin-specific peptidase6 (MER000863), ubiquitin-specific peptidase 4 (MER001795),ubiquitin-specific peptidase 8 (MER001884), ubiquitin-specific peptidase13 (MER002627), ubiquitin-specific peptidase 2 (MER004834),ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specificpeptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896),ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specificpeptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER004978),ubiquitin-specific peptidase 12 (MER005454), ubiquitin-specificpeptidase 16 (MER005493), ubiquitin-specific peptidase 15 (MER005427),ubiquitin-specific peptidase 17 (MER002900), ubiquitin-specificpeptidase 19 (MER005428), ubiquitin-specific peptidase 20 (MER005494),ubiquitin-specific peptidase 3 (MER005513), ubiquitin-specific peptidase9Y (MER004314), ubiquitin-specific peptidase 18 (MER005641),ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specificpeptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335),ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specificpeptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033),ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specificpeptidase 26 (Homo sapiens-type) (MER014292), ubiquitin-specificpeptidase 24 (MER005706), ubiquitin-specific peptidase 42 (MER011852),ubiquitin-specific peptidase 46 (MER014629), ubiquitin-specificpeptidase 37 (MER014633), ubiquitin-specific peptidase 28 (MER014634),ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specificpeptidase 38 (MER014637), ubiquitin-specific peptidase 44 (MER014638),ubiquitin-specific peptidase 50 (MER030315), ubiquitin-specificpeptidase 35 (MER014646), ubiquitin-specific peptidase 30 (MER014649),Mername-AA091 peptidase (MER014743), ubiquitin-specific peptidase 45(MER030314), ubiquitin-specific peptidase 51 (MER014769),ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specificpeptidase 48 (MER064620), ubiquitin-specific peptidase 40 (MER015483),ubiquitin-specific peptidase 41 (MER045268), ubiquitin-specificpeptidase 31 (MER015493), Mername-AA129 peptidase (MER016485),ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase(MER052579), USP17-like peptidase (MER030192), ubiquitin-specificpeptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER027329),ubiquitin-specific endopeptidase 39 [misleading] (MER064621),Mername-AA090 non-peptidase homologue (MER014739), ubiquitin-specificpeptidase 43 [misleading] (MER030140), ubiquitin-specific peptidase 52[misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene(Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase(MER014750), autophagin-2 (MER013564), autophagin-1 (MER013561),autophagin-3 (MER014316), autophagin-4 (MER064622), Cezannedeubiquitinylating peptidase (MER029042), Cezanne-2 peptidase(MER029044), tumor necrosis factor alpha-induced protein 3 (MER029050),trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase(MER152304), otubain-1 (MER029056), otubain-2 (MER029061), CylD protein(MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306),DUBA deubiquitinylating enzyme (MER086098), KIAA0459 (Homo sapiens)-likeprotein (MER122467), Otud1 protein (MER125457), glycosyltransferase 28domain containing 1, isoform CRA_c (Homo sapiens)-like (MER123606),hin1L g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3Lputative peptidase (MER115261), Josephin domain containing 1 (Homosapiens) (MER125334), Josephin domain containing 2 (Homo sapiens)(MER124068), YOD1 peptidase (MER116559), legumain (plant alpha form)(MER044591), legumain (MER001800), glycosylphosphatidylinositol:proteintransamidase (MER002479), legumain pseudogene (Homo sapiens)(MER029741), family C13 unassigned peptidases (MER175813), caspase-1(MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6(MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5(MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10(MER002579), caspase-14 (MER012083), paracaspase (MER019325),Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase(MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein(MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene(Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene(MER014766), subfamily C14A non-peptidase homologues (MER185329),subfamily C14A non-peptidase homologues (MER179956), separase (Homosapiens-type) (MER011775), separase-like pseudogene (MER014797), SENP1peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase(MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032),SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4peptidase (MER005557), pyroglutamyl-peptidase I (chordate) (MER011032),Mername-AA073 peptidase (MER029978), Sonic hedgehog protein (MER002539),Indian hedgehog protein (MER002538), Desert hedgehog protein(MER012170), dipeptidyl-peptidase III (MER004252), Mername-AA164 protein(MER020410), LOC138971 g.p. (Homo sapiens) (MER020074), Atp23 peptidase(MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N(MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase(MER001013), pyroglutamyl-peptidase II (MER012221), cytosol alanylaminopeptidase (MER002746), cystinyl aminopeptidase (MER002060),aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginylaminopeptidase-like 1 (MER012271), leukocyte-derived arginineaminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase0 (MER019730), Tata binding protein associated factor (MER026493),angiotensin-converting enzyme peptidase unit 1 (MER004967),angiotensin-converting enzyme peptidase unit 2 (MER001019),angiotensin-converting enzyme-2 (MER011061), Mername-AA153 protein(MER020514), thimet oligopeptidase (MER001737), neurolysin (MER010991),mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein(MER021317), leishmanolysin-2 (MER014492), leishmanolysin-3 (MER180031),matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8(MER001084), matrix metallopeptidase-2 (MER001080), matrixmetallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068),matrix metallopeptidase-10 (Homo sapiens-type) (MER001072), matrixmetallopeptidase-11 (MER001075), matrix metallopeptidase-7 (MER001092),matrix metallopeptidase-12 (MER001089), matrix metallopeptidase-13(MER001411), membrane-type matrix metallopeptidase-1 (MER001077),membrane-type matrix metallopeptidase-2 (MER002383), membrane-typematrix metallopeptidase-3 (MER002384), membrane-type matrixmetallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021),matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B(MER004766), membrane-type matrix metallopeptidase-5 (MER005638),membrane-type matrix metallopeptidase-6 (MER012071), matrixmetallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098),matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28(MER013587), matrix metallopeptidase-23A (MER037217), macrophageelastase homologue (chromosome 8, Homo sapiens) (MER030035),Mername-AA156 protein (MER021309), matrix metallopeptidase-like 1(MER045280), subfamily M10A non-peptidase homologues (MER175912),subfamily M10A non-peptidase homologues (MER187997), subfamily M10Anon-peptidase homologues (MER187998), subfamily M10A non-peptidasehomologues (MER180000), meprin alpha subunit (MER001111), meprin betasubunit (MER005213), procollagen C-peptidase (MER001113), mammaliantolloid-like 1 protein (MER005124), mammalian-type tolloid-like 2protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase(MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase(MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase(MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase(MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382),ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase(MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase(MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase(MER005546), ADAM28 peptidase (Homo sapiens-type) (MER005495), ADAMTS5peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase(MER005893),

ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21peptidase (Homo sapiens-type) (MER004726), ADAMTS10 peptidase(MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase(MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996),ADAMTS20 peptidase (Homo sapiens-type) (MER026906), procollagen IN-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein(MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230),ADAM32 protein (MER026938), non-peptidase homologue (Homo sapienschromosome 4) (MER029973), family M12 non-peptidase homologue (Homosapiens chromosome 16) (MER047654), family M12 non-peptidase homologue(Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homosapiens-type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein(MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267),protein similar to ADAM21 peptidase preproprotein (Homo sapiens)(MER026944), Mername-AA225 peptidase homologue (Homo sapiens)(MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens)(MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAM1 g.p. (Homosapiens) (MER003912), subfamily M12B non-peptidase homologues(MER188210), subfamily M12B non-peptidase homologues (MER188211),subfamily M12B non-peptidase homologues (MER188212), subfamily M12Bnon-peptidase homologues (MER188220), neprilysin (MER001050),endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme2 (MER004776), DINE peptidase (MER005197), neprilysin-2 (MER013406),Kell blood-group protein (MER001054), PHEX peptidase (MER002062), i-AAApeptidase (MER001246), i-AAA peptidase (MER005755), paraplegin(MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A(MER014306), pappalysin-1 (MER002217), pappalysin-2 (MER014521),farnesylated-protein converting enzyme 1 (MER002646),metalloprotease-related protein-1 (MER030873), aminopeptidase AMZ2(MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase A1(MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B(MER001194), carboxypeptidase N (MER001198), carboxypeptidase E(MER001199), carboxypeptidase M (MER001205), carboxypeptidase U(MER001193), carboxypeptidase A3 (MER001187), metallocarboxypeptidase Dpeptidase unit 1 (MER003781), metallocarboxypeptidase Z (MER003428),metallocarboxypeptidase D peptidase unit 2 (MER004963), carboxypeptidaseA4 (MER013421), carboxypeptidase A6 (MER013456), carboxypeptidase A5(MER017121), metallocarboxypeptidase 0 (MER016044), cytosoliccarboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosoliccarboxypeptidase 1 (MER033179), cytosolic carboxypeptidase 2(MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964),adipocyte-enhancer binding protein 1 (MER003889), carboxypeptidase-likeprotein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764),cytosolic carboxypeptidase (MER026952), family M14 non-peptidasehomologues (MER199530), insulysin (MER001214), mitochondrial processingpeptidase beta-subunit (MER004497), nardilysin (MER003883), eupitrilysin(MER004877), mitochondrial processing peptidase non-peptidase alphasubunit (MER001413), ubiquinol-cytochrome c reductase core protein I(MER003543), ubiquinol-cytochrome c reductase core protein II(MER003544), ubiquinol-cytochrome c reductase core protein domain 2(MER043998), insulysin unit 2 (MER046821), nardilysin unit 2(MER046874), insulysin unit 3 (MER078753), mitochondrial processingpeptidase subunit alpha unit 2 (MER124489), nardilysin unit 3(MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), subfamily M16Bnon-peptidase homologues (MER188757), leucyl aminopeptidase (animal)(MER003100), Mername-AA040 peptidase (MER003919), leucylaminopeptidase-1 (Caenorhabditis-type) (MER013416), methionylaminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728),aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote)(MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediatecleaving peptidase 55 kDa (MER013463), mitochondrial methionylaminopeptidase (MER014055), Mername-AA020 peptidase homologue(MER010972), proliferation-association protein 1 (MER005497),chromatin-specific transcription elongation factor 140 kDa subunit(MER026495), proliferation-associated protein 1-like (Homo sapienschromosome X) (MER029983), Mername-AA226 peptidase homologue (Homosapiens) (MER056262), Mername-AA227 peptidase homologue (Homo sapiens)(MER047299), subfamily M24A non-peptidase homologues (MER179893),aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase(MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidaseI (MER015142), Mername-AA161 protein (MER021873), aminoacylase(MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE Lpeptidase (MER005239), glutamate carboxypeptidase III (MER005238),plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase(MER015091), Fxna peptidase (MER029965), transferrin receptor protein(MER002105), transferrin receptor 2 protein (MER005152), glutaminylcyclise (MER015095), glutamate carboxypeptidase II (Homo sapiens)-typenon-peptidase homologue (MER026971), nicalin (MER044627), membranedipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499),membrane-bound dipeptidase-3 (MER013496), dihydro-orotase (MER005767),dihydropyrimidinase (MER033266), dihydropyrimidinase related protein-1(MER030143), dihydropyrimidinase related protein-(MER030155),dihydropyrimidinase related protein-3 (MER030151), dihydropyrimidinaserelated protein-4 (MER030149), dihydropyrimidinase related protein-5(MER030136), hypothetical protein like 5730457F11RIK (MER033184),1300019j08rik protein (MER033186)), guanine aminohydrolase (MER037714),Keel putative peptidase (MER001577), OSGEPL1-like protein (MER013498),S2P peptidase (MER004458), subfamily M23B non-peptidase homologues(MER199845), subfamily M23B non-peptidase homologues (MER199846),subfamily M23B non-peptidase homologues (MER199847), subfamily M23Bnon-peptidase homologues (MER137320), subfamily M23B non-peptidasehomologues (MER201557), subfamily M23B non-peptidase homologues(MER199417), subfamily M23B non-peptidase homologues (MER199418),subfamily M23B non-peptidase homologues (MER199419), subfamily M23Bnon-peptidase homologues (MER199420), subfamily M23B non-peptidasehomologues (MER175932), subfamily M23B non-peptidase homologues(MER199665), Poh1 peptidase (MER020382), Jab1/MPN domain metalloenzyme(MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase(MER021890), histone H2A deubiquitinase MYSM1 (MER021887), AMSHdeubiquitinating peptidase (MER030146), putative peptidase (Homo sapienschromosome 2) (MER029970), Mername-AA168 protein (MER021886), COP9signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatorysubunit 7 (MER030134), eukaryotic translation initiation factor 3subunit 5 (MER030133), IFP38 peptidase homologue (MER030132), subfamilyM67A non-peptidase homologues (MER191181), subfamily M67A unassignedpeptidases (MER191144), granzyme B (Homo sapiens-type) (MER000168),testisin (MER005212), tryptase beta (MER000136), kallikrein-relatedpeptidase 5 (MER005544), corin (MER005881), kallikrein-related peptidase12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1(MER011036), kallikrein-related peptidase 14 (MER011038),hyaluronan-binding peptidase (MER003612), transmembrane peptidase,serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130),adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homosapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119),tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembranepeptidase, serine 3 (MER005926), kallikrein-related peptidase 15(MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase(MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204peptidase (MER029980), cationic trypsin (Homo sapiens-type) (MER000020),elastase-2 (MER000118), mannan-binding lectin-associated serinepeptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin(MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase(Homo sapiens-type) (MER000123), tryptase alpha (MER000135), granzyme K(MER001936), granzyme H (MER000166), chymotrypsin B (MER000001),elastase-1 (MER003733), pancreatic endopeptidase E (MER000149),pancreatic elastase II (MER000146), enteropeptidase (MER002068),chymotrypsin C (MER000761), prostasin (MER002460), kallikrein 1(MER000093), kallikrein-related peptidase 2 (MER000094),kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022),complement component C1r-like peptidase (MER016352), complement factor D(MER000130), complement component activated C1r (MER000238), complementcomponent activated C1s (MER000239), complement component C2a(MER000231), complement factor B (MER000229), mannan-bindinglectin-associated serine peptidase 1 (MER000244), complement factor I(MER000228), pancreatic endopeptidase E form B (MER000150), pancreaticelastase IIB (MER000147), coagulation factor XIIa (MER000187), plasmakallikrein (MER000203) coagulation factor Xia (MER000210), coagulationfactor IXa (MER000216), coagulation factor Vila (MER000215), coagulationfactor Xa (MER000212), thrombin (MER000188), protein C (activated)(MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growthfactor activator (MER000186), mannan-binding lectin-associated serinepeptidase 2 (MER002758), u-plasminogen activator (MER000195),t-plasminogen activator (MER000192), plasmin (MER000175),kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171),kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4(MER005266), prosemin (MER004214),

chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861),kallikrein-related peptidase 11 (MER216142), trypsin-2 type A(MER000021), HtrA1 peptidase (Homo sapiens-type) (MER002577), HtrA2peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase(MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461),TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2(MER021884), trypsin C (MER021898), kallikrein-related peptidase 7(MER002001), matriptase (MER003735), kallikrein-related peptidase 13(MER005269), kallikrein-related peptidase 9 (MER005270), matriptase-2(MER005278), umbelical vein peptidase (MER005421), LCLP peptidase(MER001900), spinesin (MER014385), marapsin-2 (MER021929), complementfactor D-like putative peptidase (MER056164), ovochymase-2 (MER022410),HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412),epidermis-specific SP-like putative peptidase (MER029900), testis serinepeptidase 5 (MER029901), Mername-AA258 peptidase (MER000285),polyserase-IA unit 1 (MER030879), polyserase-IA unit 2 (MER030880),testis serine peptidase 2 (human-type) (MER033187), hypotheticalacrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase(MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2(MER061748), peptidase similar to tryptophan/serine protease(MER056263), polyserase-2 unit 1 (MER061777), Mername-AA123 peptidase(MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein(MER099172), hCG22067 (Homo sapiens) (MER099169), brain-rescue-factor-1(Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173),polyserase-2 unit 2 (MER061760), polyserase-2 unit 3 (MER065694),Mername-AA201 (peptidase homologue) MER099175, secreted trypsin-likeserine peptidase homologue (MER030000), polyserase-1A unit 3(MER029880), azurocidin (MER000119), haptoglobin-1 (MER000233),haptoglobin-related protein (MER000235), macrophage-stimulating protein(MER001546), hepatocyte growth factor (MER000185), protein Z(MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132),plasma kallikrein-like protein 4 (MER016346), PRSS35 protein(MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein(MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), tryptasepseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptasepseudogene III (MER015079), subfamily S1A unassigned peptidases(MER216982), subfamily S1A unassigned peptidases (MER216148),amidophosphoribosyltransferase precursor (MER003314),glutamine-fructose-6-phosphate transaminase 1 (MER003322),glutamine:fructose-6-phosphate amidotransferase (MER012158),Mername-AA144 protein (MER021319), asparagine synthetase (MER033254),family C44 non-peptidase homologues (MER159286), family C44 unassignedpeptidases (MER185625) family C44 unassigned peptidases (MER185626),secernin 1 (MER045376), secernin 2 (MER064573), secernin 3 (MER064582),acid ceramidase precursor (MER100794), N-acylethanolamine acid amidaseprecursor (MER141667), proteasome catalytic subunit 1 (MER000556),proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit3 (MER002149), proteasome catalytic subunit 1i (MER000552), proteasomecatalytic subunit 2i (MER001515), proteasome catalytic subunit 3i(MER000555), proteasome catalytic subunit 5t (MER026203), protein serinekinase c17 (MER026497), proteasome subunit alpha 6 (MER000557),proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4(MER000554), proteasome subunit alpha 7 (MER033250), proteasome subunitalpha 5 (MER000558), proteasome subunit alpha 1 (MER000549), proteasomesubunit alpha 3 (MER000553), proteasome subunit XAPC7 (MER004372),proteasome subunit beta 3 (MER001710), proteasome subunit beta 2(MER002676), proteasome subunit beta 1 (MER000551), proteasome subunitbeta 4 (MER001711), Mername-AA230 peptidase homologue (Homo sapiens)(MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172),Mername-AA232 pseudogene (Homo sapiens) (MER047316),glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase(threonine type) (MER031622), taspase-1 (MER016969),gamma-glutamyltransferase 5 (mammalian-type) (MER001977),gamma-glutamyltransferase 1 (mammalian-type) (MER001629),gamma-glutamyltransferase 2 (Homo sapiens) (MER001976),gamma-glutamyltransferase-like protein 4 (MER002721),gamma-glutamyltransferase-like protein 3 (MER016970), similar togamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204),similar to gamma-glutamyltransferase 1 precursor (Homo sapiens)(MER026205), Mername-AA211 putative peptidase (MER026207),gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidasehomologue (chromosome 2, Homo sapiens) (MER037241), polycystin-1(MER126824), KIAA1879 protein (MER159329), polycystic kidney disease1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine5″-monophosphate synthetase (MER043387), carbamoyl-phosphate synthase(Homo sapiens-type) (MER078640), dihydro-orotase (N-terminal unit) (Homosapiens-type) (MER060647), DJ-1 putative peptidase (MER003390),Mername-AA100 putative peptidase (MER014802), Mername-AA101non-peptidase homologue (MER014803), KIAA0361 protein (Homosapiens-type) (MER042827), FLJ34283 protein (Homo sapiens) (MER044553),non-peptidase homologue chromosome 21 open reading frame 33 (Homosapiens) (MER160094), family C56 non-peptidase homologues (MER177016),family C56 non-peptidase homologues (MER176613), family C56non-peptidase homologues (MER176918), EGF-like module containingmucin-like hormone receptor-like 2 (MER037230), CD97 antigen (humantype) (MER037286), EGF-like module containing mucin-like hormonereceptor-like 3 (MER037288), EGF-like module containing mucin-likehormone receptor-like 1 (MER037278), EGF-like module containingmucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAGseven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64(Mus musculus)-type protein (MER123205), GPR56 (Homo sapiens)-typeprotein (MER122057), latrophilin 2 (MER122199), latrophilin-1(MER126380), latrophilin 3 (MER124612), protocadherin Flamingo 2(MER124239), ETL protein (MER126267), G protein-coupled receptor 112(MER126114), seven transmembrane helix receptor (MER125448), Gpr114protein (MER159320), GPR126 vascular inducible G protein-coupledreceptor (MER140015), GPR125 (Homo sapiens)-type protein (MER159279),GPR116 (Homo sapiens)-type G-protein coupled receptor (MER159280),GPR128 (Homo sapiens)-type G-protein coupled receptor (MER162015),GPR133 (Homo sapiens)-type protein (MER159334), GPR110 G-protein coupledreceptor (MER159277), GPR97 protein (MER159322), KPG_006 protein(MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335),unassigned homologue (MER166269), GPR113 protein (MER159352),brain-specific angiogenesis inhibitor 2 (MER159746), PIDDauto-processing protein unit 1 (MER020001), PIDD auto-processing proteinunit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan(MER054741), proprotein convertase 9 (MER022416), site-1 peptidase(MER001948), furin (MER000375), proprotein convertase 1 (MER000376),proprotein convertase 2 (MER000377), proprotein convertase 4(MER028255), PACE4 proprotein convertase (MER000383), proproteinconvertase 5 (MER002578), proprotein convertase 7 (MER002984),tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidasehomologues (MER201339), subfamily S8A non-peptidase homologues(MER191613), subfamily S8A unassigned peptidases (MER191611), subfamilyS8A unassigned peptidases (MER191612), subfamily S8A unassignedpeptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyloligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote)(MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activationprotein alpha subunit (MER000399), PREPL A protein (MER004227),dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923),FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase(MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196putative peptidase (MER017368), Mername-AA197 putative peptidase(MER017371), C14orf29 protein (MER033244), hypothetical protein(MER033245), hypothetical esterase/lipase/thioesterase (MER047309),protein bat5 (MER037840), hypothetical protein 940219 (MER033212),hypothetical protein 937464 (MER033240), hypothetical protein 933678(MER033241), dipeptidylpeptidase homologue DPP6 (MER000403),dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Musmusculus chromosome 20 open reading frame 135 (MER037845), kynurenineformamidase (MER046020), thyroglobulin precursor (MER011604),acetylcholinesterase (MER033188), cholinesterase (MER033198),carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220),carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bilesalt-dependent lipase (MER033227), carboxylesterase-related protein(MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked(MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126),arylacetamide deacetylase (MER033237), KIAA1363-like protein(MER033242), hormone-sensitive lipase (MER033274), neuroligin 1(MER033280), neuroligin 2 (MER033283), family S9 non-peptidasehomologues (MER212939), family S9 non-peptidase homologues (MER211490),subfamily S9C unassigned peptidases (MER192341), family S9 unassignedpeptidases (MER209181), family S9 unassigned peptidases (MER200434),family S9 unassigned peptidases (MER209507), family S9 unassignedpeptidases (MER209142), serine carboxypeptidase A (MER000430),vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase(MER010960), family S15 unassigned peptidases (MER199442), family S15unassigned peptidases (MER200437), family S15 unassigned peptidases(MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446),dipeptidyl-peptidase 11 (MER004952), thymus-specific serine peptidase(MER005538), epoxide hydrolase-like putative peptidase (MER031614),Loc328574-like protein (MER033246), abhydrolase domain-containingprotein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specifictranscript protein (MER199890), mesoderm specific transcript protein(MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxidehydrolase (MER213866), similar to hypothetical protein FLJ22408(MER031608), CGI-58 putative peptidase (MER030163), Williams-Beurensyndrome critical region protein 21 epoxide hydrolase (MER031610),epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxidehydrolase) (MER031617), monoglyceride lipase (MER033247), hypotheticalprotein (MER033249), valacyclovir hydrolase (MER033259),Ccg1-interacting factor b (MER210738), glycosylasparaginase precursor(MER003299), isoaspartyl dipeptidase (threonine type) (MER031622).taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian-type)(MER001977), gamma-glutamyltransferase 1 (mammalian-type) (MER001629),gamma-glutamyltransferase 2 (Homo sapiens) (MER001976),gamma-glutamyltransferase-like protein 4 (MER002721).gamma-glutamyltransferase-like protein 3 (MER016970). similar togamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204).similar to gamma-glutamyltransferase 1 precursor (Homo sapiens)(MER026205). Mername-AA211 putative peptidase (MER026207).gamma-glutamyltransferase 6 (MER159283). gamma-glutamyl transpeptidasehomologue (chromosome 2, Homo sapiens) (MER037241). polycystin-1(MER126824), KIAA1879 protein (MER159329). polycystic kidney disease1-like 3 (MER172554). gamma-glutamyl hydrolase (MER002963). guanine5″-monophosphate synthetase (MER043387). carbamoyl-phosphate synthase(Homo sapiens-type) (MER078640). dihydro-orotase (N-terminal unit) (Homosapiens-type) (MER060647). DJ-1 putative peptidase (MER003390).Mername-AA100 putative peptidase (MER014802). Mername-AA101non-peptidase homologue (MER014803). KIAA0361 protein (Homosapiens-type) (MER042827). FLJ34283 protein (Homo sapiens) (MER044553).non-peptidase homologue chromosome 21 open reading frame 33 (Homosapiens) (MER160094). family C56 non-peptidase homologues (MER177016),family C56 non-peptidase homologues (MER176613). family C56non-peptidase homologues (MER176918). EGF-like module containingmucin-like hormone receptor-like 2 (MER037230). CD97 antigen (humantype) (MER037286). EGF-like module containing mucin-like hormonereceptor-like 3 (MER037288). EGF-like module containing mucin-likehormone receptor-like 1 (MER037278). EGF-like module containingmucin-like hormone receptor-like 4 (MER037294). cadherin EGF LAGseven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64(Mus musculus)-type protein (MER123205). GPR56 (Homo sapiens)-typeprotein (MER122057). latrophilin 2 (MER122199). latrophilin-1(MER126380). latrophilin 3 (MER124612). protocadherin Flamingo 2(MER124239). ETL protein (MER126267). G protein-coupled receptor 112(MER126114). seven transmembrane helix receptor (MER125448). Gpr114protein (MER159320). GPR126 vascular inducible G protein-coupledreceptor (MER140015). GPR125 (Homo sapiens)-type protein (MER159279).GPR116 (Homo sapiens)-type G-protein coupled receptor (MER159280).GPR128 (Homo sapiens)-type G-protein coupled receptor (MER162015).GPR133 (Homo sapiens)-type protein (MER159334) GPR110 G-protein coupledreceptor (MER159277), GPR97 protein (MER159322), KPG_006 protein(MER161773) KPG_008 protein (MER161835), KPG_009 protein (MER159335),unassigned homologue (MER166269), GPR113 protein (MER159352),brain-specific angiogenesis inhibitor 2 (MER159746), PIDDauto-processing protein unit 1 (MER020001), PIDD auto-processing proteinunit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan(MER054741), proprotein convertase 9 (MER022416), site-1 peptidase(MER001948), furin (MER000375), proprotein convertase 1 (MER000376),proprotein convertase 2 (MER000377), proprotein convertase 4(MER028255), PACE4 proprotein convertase (MER000383), proproteinconvertase 5 (MER002578), proprotein convertase 7 (MER002984),tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidasehomologues (MER201339), subfamily S8A non-peptidase homologues(MER191613), subfamily S8A unassigned peptidases (MER191611), subfamilyS8A unassigned peptidases (MER191612), subfamily S8A unassignedpeptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyloligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote)(MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activationprotein alpha subunit (MER000399), PREPL A protein (MER004227),dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923),FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase(MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196putative peptidase (MER017368), Mername-AA197 putative peptidase(MER017371), C14orf29 protein (MER033244), hypothetical protein(MER033245), hypothetical esterase/lipase/thioesterase (MER047309),protein bat5 (MER037840), hypothetical protein 940219 (MER033212),hypothetical protein 937464 (MER033240), hypothetical protein 933678(MER033241), dipeptidylpeptidase homologue DPP6 (MER000403),dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Musmusculus chromosome 20 open reading frame 135 (MER037845), kynurenineformamidase (MER046020), thyroglobulin precursor (MER011604),acetylcholinesterase (MER033188), cholinesterase (MER033198),carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220),carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bilesalt-dependent lipase (MER033227), carboxylesterase-related protein(MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked(MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126),arylacetamide deacetylase (MER033237), KIAA1363-like protein(MER033242), hormone-sensitive lipase (MER033274), neuroligin 1(MER033280), neuroligin 2 (MER033283), family S9 non-peptidasehomologues (MER212939), family S9 non-peptidase homologues (MER211490),subfamily S9C unassigned peptidases (MER192341), family S9 unassignedpeptidases (MER209181), family S9 unassigned peptidases (MER200434),family S9 unassigned peptidases (MER209507), family S9 unassignedpeptidases (MER209142), serine carboxypeptidase A (MER000430),vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase(MER010960), family S15 unassigned peptidases (MER199442), family S15unassigned peptidases (MER200437), family S15 unassigned peptidases(MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446),dipeptidyl-peptidase 11 (MER004952), thymus-specific serine peptidase(MER005538), epoxide hydrolase-like putative peptidase (MER031614),Loc328574-like protein (MER033246), abhydrolase domain-containingprotein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specifictranscript protein (MER199890), mesoderm specific transcript protein(MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxidehydrolase (MER213866), similar to hypothetical protein FLJ22408(MER031608), CGI-58 putative peptidase (MER030163), Williams-Beurensyndrome critical region protein 21 epoxide hydrolase (MER031610),epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxidehydrolase) (MER031617), monoglyceride lipase (MER033247), hypotheticalprotein (MER033249), valacyclovir hydrolase (MER033259),Ccg1-interacting factor b (MER210738).

It will be appreciated that for a given unwanted cell type, the skilledperson can readily determine an appropriate one or more proteasecleavage sites to use, for example by consulting scientific literatureto determine which proteases are overexpressed by that cell type.Oncomine (https://www.oncomine.org) is an online cancer gene expressiondatabase, and so when the molecule of the invention is for treatingcancer, the skilled person may search the Oncomine database to identifya particular one or more protease cleavage sites that will beappropriate for treating a given cancer type. Alternative databasesinclude European Bioinformatic Institute (http://www.ebi.ac.uk) inparticular (http://www.ebi.ac.uk/gxa). Protease databases include PMAP(http://www.proteolysis.org), ExPASy Peptide Cutter(http://ca.expasy.org/tools/peptide cutter) and PMAP.Cut DB(http://cutdb.burnham.org).

It is noted that it may be desirable to screen a library of peptidesincorporating multiple potential cleavage sites and evaluating theoptimal cleavage site for a given unwanted cell (eg tumour). Suchpeptides may be useful as linkers, for example, to join the furthermoiety to one or more masking moieties, or to promote a particularconformation of the further moiety so as to mask T cell binding regionof the further moiety (see embodiment exemplified by FIG. 1).

TABLE 4 Matrix showing preferred protease cleavage sites for treatingparticular tumours Lung- Protease Substrate Breast Ovarian EndometrialCervical Bladder Renal Melanoma NSLC Lung-SLC Prostate TesticularThyroid Brain Serine urokinase-type uPA CPGR-VVGG • • • • • • • •plasminogen (SEQ ID No: 1) activator tPA CPGR-VVGG • (SEQ ID No: 1)Cathepsin A • Cathepsin G Plasmin GGR-X (SEQ ID No: 2) C1s YLGR-SYKV(SEQ No: 3) or MQLGR-X (SEQ ID No: 4) MASP2 SLGR-KIQI (SEQ ID No: 5)Thrombin LVPRGS (SEQ ID No: 6) Trypsin XXXR-X (SEQ ID No: 7)Chymotrypsin Elastase 1 Leucocyte/Neut Elastase 2 AAPV-X (SEQ ElastaseID No: 8) Leucocyte/Neut Elastase 2 AAPV-X (SEQ Elastase ID No: 8)MT-SP1/ST14 Matriptase KQLR-VVNG • • • (SEQ ID No: 9) or KQSR- (SEQ IDNo: 10) KFVP (SEQ ID No: 11) MT-SP2 Matriptase2 • TMPRSS1 Hepsin • • •TMPRSS2 GGR-X (SEQ • ID No: 2) TMPRSS3 TMPRSS4 PSA Prostate SpecificSSKYQ (SEQ • Antigen ID No: 12) or HSSKLQL (SEQ ID No: 13)Leucocyte/Neut Elastase 2 AAPV-X (SEQ Elastase ID No: 8) MT-SP1/ST14Matriptase KQLR-VVNG • • • (SEQ ID No: 9) MT-SP2 Matriptase2 • TMPRSS1Hepsin • • • TMPRSS2 GGR-X (SEQ • ID No: 2) TMPRSS3 TMPRSS4 PSA ProstateSpecific SSKYQ (SEQ • Antigen ID No: 12) or HSSKLQL (SEQ ID No: 13)Cysteine Cathepsin B GGGG-F (SEQ • • • • ID No: 14) Cathepsin LCathepsin F Cathepsin H • Cathepsin K • • • Cathpsin L1 Cathepsin L2Cathepsin O • Cathepsin W Cathepsin S • Cathepsin Z (or X) AsparticCathepsin D • • • Cathepsin E Metallo Collagenase 1 MMP1 PLG-LLG • • • •(SEQ ID No: 15) Gelatinase A MMP2 PQG-IAGQ • • • • • (SEQ ID No: 16) orPVGLIG (SEQ ID No: 17) Stromelysin MMP3 • MMP4 MMP5 MMP6 Matrilysin MMP7Collagenase 2 MMP8 Gelatinase B MMP9 PQG-IAGQ • • • (SEQ ID No: 16) orPRA-LY (SEQ ID No: 18) MMP10 MMP11 • MMP12 • MMP13 MMP14 PRH-LR (SEQ • •• • • • • • ID No: 19) MMP15 • • • MMP16 MMP17 • MMP18 MMP19 MMP20 MMP21MMP23A MMP23B MMP24 • MMP25 MMP26 • • MMP27 MMP28 • ADAM2 ADAM7 ADAM8 •• • • • ADAM9 • • • ADAM10 • • • Metallo ADAM11 ADAM12 • • • ADAM15 • •• • ADAM17 • • • • • • ADAM18/27 ADAM19 • • ADAM20 ADAM21/31 ADAM22ADAM23 ADAM28 • • ADAM29 ADAM30 ADAM33 ADAMTS1 ADAMTS2 ADAMTS3 ADAMTS4ADAMTS5/11 ADAMTS6 ADAMTS7 ADAMTS8 ADAMTS9 ADAMTS10 ADAMTS12 ADAMTS13ADAMTS14 ADAMTS15 • ADAMTS16 ADAMTS17 ADAMTS18 ADAMTS19 ADAMTS20Protease Substrate Oesophageal Gastric Pancreatic Colorectal LiverLeukaemia Myeloma NHL Hodgkin's AML ALL CLL Model Cell line Serineurokinase-type uPA CPGR-VVGG • • • • • • • • • • plasminogen (SEQ IDNo: 1) activator tPA CPGR-VVGG (SEQ ID No: 1) Cathepsin A Cathepsin GPlasmin GGR-X (SEQ ID No: 2) C1s YLGR-SYKV (SEQ No: 3) or MQLGR-X (SEQID No: 4) MASP2 SLGR-KIQI (SEQ ID No: 5) Thrombin LVPRGS (SEQ ID No: 6)Trypsin XXXR-X (SEQ ID No: 7) Chymotrypsin Elastase 1 Leucocyte/NeutElastase 2 AAPV-X (SEQ Elastase ID No: 8) Leucocyte/Neut Elastase 2AAPV-X (SEQ Elastase ID No: 8) MT-SP1/ST14 Matriptase KQLR-VVNG • (SEQID No: 9) or KQSR- (SEQ ID No: 10) KFVP (SEQ ID No: 11) MT-SP2Matriptase2 TMPRSS1 Hepsin TMPRSS2 GGR-X (SEQ ID No: 2) TMPRSS3 TMPRSS4• • • PSA Prostate Specific SSKYQ (SEQ Antigen ID No: 12) or HSSKLQL(SEQ ID No: 13) Leucocyte/Neut Elastase 2 AAPV-X (SEQ Elastase ID No: 8)MT-SP1/ST14 Matriptase KQLR-VVNG • (SEQ ID No: 9) MT-SP2 Matriptase2TMPRSS1 Hepsin TMPRSS2 GGR-X (SEQ ID No: 2) TMPRSS3 TMPRSS4 • • • PSAProstate Specific SSKYQ (SEQ Antigen ID No: 12) or HSSKLQL (SEQ ID No:13) Cysteine Cathepsin B GGGG-F (SEQ ID No: 14) Cathepsin L Cathepsin FCathepsin H Cathepsin K Cathpsin L1 Cathepsin L2 Cathepsin O Cathepsin WCathepsin S Cathepsin Z (or X) Aspartic Cathepsin D • Cathepsin EMetallo Collagenase 1 MMP1 PLG-LLG • • • • • PMA (SEQ ID No: Activated15) U937 and MCF7 cells, MDA-MB231 Gelatinase A MMP2 PQG-IAGQ • • • •Colo205, HT29 (SEQ ID No: 16) or PVGLIG (SEQ ID No: 17) Stromelysin MMP3MMP4 MMP5 MMP6 Matrilysin MMP7 • • • Collagenase 2 MMP8 Gelatinase BMMP9 PQG-IAGQ • MCF7, PC3 (SEQ ID No: 16) or PRA-LY (SEQ ID No: 18)MMP10 MMP11 MMP12 MMP13 MMP14 PRH-LR (SEQ • • • • ID No: 19) MMP15 MMP16MMP17 MMP18 MMP19 THP-1, HL-60 MMP20 MMP21 MMP23A MMP23B • MMP24 MMP25MMP26 MMP27 MMP28 • • • ADAM2 ADAM7 ADAM8 • ADAM9 • • • ADAM10 • • •Metallo ADAM11 ADAM12 • • • ADAM15 • ADAM17 • • • • LNCaP, MDA-MB231,MCF7 express ADAM17. Colo205 express barely any ADAM18/27 ADAM19 ADAM20ADAM21/31 ADAM22 ADAM23 ADAM28 ADAM29 ADAM30 ADAM33 ADAMTS1 ADAMTS2ADAMTS3 ADAMTS4 ADAMTS5/11 ADAMTS6 ADAMTS7 ADAMTS8 • ADAMTS9 ADAMTS10ADAMTS12 ADAMTS13 ADAMTS14 ADAMTS15 ADAMTS16 ADAMTS17 ADAMTS18 ADAMTS19ADAMTS20

TABLE 5 Tumour sites in which ADAM overexpression has been reportedProtein Tumour expression ADAM8 Brain, kidney, lung, pancreas ADAM9Breast, gastric, liver, lung, pancreas, prostate ADAM10 Colon, gastric,leukaemia, prostate, uterus, ovary ADAM12 Bladder, brain, breast, colon,gastric, liver ADAM15 Breast, gastric, lung, prostate ADAM17 Brain,breast, colon, gastric, kidney, liver, lung, ovary, pancreas, prostateADAM19 Brain, kidney ADAM28 Breast, kidney, lung

A number of the proteolytic ADAMs (a disintegrin and metalloproteinase)have been detected in cancers and mRNA or protein levels have been foundto be upregulated relative to normal tissue (adapted from Nature ReviewsCancer 8, 932-941 (December 2008)|doi:10.1038/nrc2459).

In one embodiment, the protease may be an esterase.

Other cleavage sites include linkages which are labile under certainconditions in the vicinity of unwanted cells (eg tumourmicroenvironment). For example, the one or more cleavage sites maycomprise disulphide bonds, which can be reduced in the hypoxic tumourmicroenvironment, or may comprise pH sensitive moieties that break inacidic conditions. It will be understood, however, that the one or morecleavage sites must be selectively cleavable in the vicinity of theunwanted cells and so such linkages must be more labile and preferablyonly labile in the vicinity of unwanted cells compared to in thevicinity of wanted cells.

Alternatively, the one or more cleavage sites may comprise nucleic acid(eg DNA or RNA) that is selectively cleavable in the vicinity ofunwanted cells (eg by nucleases). Other cleavage sites includephosphate, lipid or disulphide containing moieties that may be cleavableby appropriate enzymes.

Synthesis of Molecule of Invention

Conveniently, the targeting moiety is joined to the further moiety byone or more linkers. Thus, by ‘linker’ we include the meaning of achemical moiety that attaches the targeting moiety to the furthermoiety. Preferably, the linker is a peptide linker. It will beappreciated that such linkers may contain one or more cleavage sitesthat are selectively cleaved in the vicinity of the unwanted cells so asto unmask the immune cell binding region of the further moiety.

As described above, it is possible for the further moiety to have two ormore separate parts. For example, the further moiety may comprise two ormore polypeptide domains including immunoglobulin domains of an antibody(e.g. scFv antibody). Such parts may be linked together by one or morelinkers. Thus, by linker′, we also include the meaning of any chemicalmoiety that attaches the separate parts of a further moiety to eachother. Preferably, the linker is a peptide linker. For example, thefurther moiety of the molecule illustrated in FIG. 1 comprises a VHdomain and a VL domain which domains are joined by a linker. Theselinkers may contain one or more cleavage sites that are selectivelycleaved in the vicinity of the unwanted cells so as to unmask the T cellbinding region of the further moiety

Also conveniently, when the molecule comprises one or more maskingmoieties, these are attached to the targeting moiety and/or furthermoiety by means of one or more linkers. Thus, by ‘linker’ we alsoinclude the meaning of a chemical moiety that attaches the targetingmoiety to a masking moiety and a chemical moiety that attaches thefurther moiety to a masking moiety. Preferably, the linker is a peptidelinker. For example, FIGS. 2 and 3 depict examples of linkers between afurther moiety and a masking moiety, and FIG. 4 depicts examples oflinkers between a further moiety and a masking moiety, and between atargeting moiety and a masking moiety. Such linkers may also contain oneor more cleavage sites that are selectively cleaved in the vicinity ofthe unwanted cells so as to unmask the T cell binding region of thefurther moiety

Examples of suitable linkers include peptides, polymers, nucleotides,nucleic acids, polysaccharides and lipid organic species (egpolyethylene glycol). Most preferably, however, the linkers describedherein are peptide linkers. Generally, the peptide linkers have between2 and 100 amino acid residues, such as more than or less than 10, 20,30, 40, 50, 60, 70, 80 and 90 amino acids. Further detail of thepreferred length of particular linkers is given below.

It is appreciated that the targeting moiety may either be boundcovalently or non-covalently to the further moiety. Likewise, it isappreciated that the targeting moiety and/or further moiety may eitherbe bound covalently or non-covalently to the one or more maskingmoieties.

Typically, the targeting moiety is covalently bound to the furthermoiety, and when the molecule comprises one or more masking moieties,the one or more masking moieties are covalently bound to the furthermoiety and/or targeting moiety.

In one embodiment, the targeting moiety and further moiety arecovalently attached by a linker (eg peptide linker). When the moleculecomprises one or more masking moieties, the one or more masking moietiesmay be covalently attached to the further moiety and/or targeting moietyby one or more linkers (eg peptide linkers).

It is appreciated that the targeting moiety, further moiety (includingseparate parts thereof) and one or more masking moieties do not need tobe linked directly to each other, but may be attached via one or morespacer moieties (eg peptides). For example, the targeting moiety may belinked to a chemical moiety (eg peptide) which in turn is linked to thefurther moiety. Similarly, the further moiety may be linked to one ormore chemical moieties (eg peptides) which in turn are linked to one ormore respective masking moieties. In one embodiment, such spacermoieties may comprise a cleavage site that is cleavable selectively inthe vicinity of the unwanted cells, as discussed above. It will beappreciated that the spacer moiety may serve to prevent steric hindranceand facilitate protease cleavage.

In view of the above, it is appreciated that the invention provides amolecule comprising: (i) a targeting moiety capable of directly orindirectly targeting to unwanted cells, (ii) a further moiety that has amasked immune cell binding region so as to prevent binding of thefurther moiety to an immune cell, (iii) one or more masking moietiesattached to the further moiety and/or targeting moiety which one or moremasking moieties act to mask the immune cell binding region of thefurther moiety, and (iv) one or more cleavable sites between the one ormore masking moieties, and the further moiety and/or the targetingmoiety, wherein the one or more cleavage sites can be selectivelycleaved in the vicinity of the unwanted cells so as to unmask the immunecell binding region.

In a preferred embodiment where the targeting moiety, further moietyand, if present, the one or more masking moieties are covalentlyattached and where all moieties are peptides or polypeptides, it isappreciated that the component moieties of the molecule may be part ofone or more fusion polypeptides that may be encoded by a respective oneor more nucleic acid molecules. The invention includes such a nucleicacid molecules and host immune cells containing them. For example, atargeting moiety (eg antibody) may be genetically engineered to containthe further moiety and, if present, the one or more masking moieties,using genetic engineering techniques well established in the art. Thus,it will be appreciated that the further moiety (including separate partsthereof) and, if present, the one or more masking moieties, may beembedded within the polypeptide sequence of the targeting moiety,provided that the immune cell binding region of the further moiety canbe unmasked when the molecule is in the vicinity of unwanted cells, suchthat the further moiety can bind to an immune cell. It will beappreciated that the each of the targeting moiety, further moiety and,if present, one or more masking moieties need not be encoded bycontiguous sections of polynucleotide. For example, although the furthermoiety may be fused to the terminus of the targeting moiety, and, ifpresent, the one or more masking moieties fused to the terminus of thefurther moiety, it is also contemplated that the various moieties of themolecule may be encoded by non-contiguous polynucleotide sequences, andbetween these non-contiguous polynucleotide sequences, polynucleotidessequences may exist that encode other moieties of the molecule. Forexample, it is clear from FIG. 4 that separate parts of the furthermoiety are encoded by non-contiguous sections of polynucleotide sequenceand between these sections there is a polynucleotide sequence thatencodes the targeting moiety.

It is further appreciated that the molecule of the invention maycomprise two or more polypeptide chains that fold into the correctconformation where the two or more polypeptide chains are mixed. Each ofthe two or more polypeptides may include one or more of the targetingmoiety, further moiety (including separate parts thereof) and, ifpresent, the one or more masking moieties. This is illustrated by themolecule depicted in FIG. 2 and is particularly relevant when any of thetargeting moiety, further moiety and, if present, the one or moremasking moieties comprise one or more immunoglobulin domains, since thedomains on one polypeptide chain (eg VH domain) may pair with domains onanother polypeptide chain (eg VL domain). Pairing of immunoglobulindomains derived from different polypeptide chains is well known in theart, and forms the basis of the preparation of diabodies and triabodies.

It follows that when in the specific embodiment where the targetingmoiety, further moiety and, if present, the one or more masking moietiesare covalently attached and where all moieties are peptides orpolypeptides, the molecule of the invention may be considered to be abispecific antibody (eg a BITE bispecific antibody). For example, thebispecific antibody may comprise (i) a first portion that is capable ofspecifically binding to an unwanted cell and (ii) a second portion thatcomprises a masked immune cell binding region so as to prevent bindingof the second portion to an immune cell, wherein the masked immune cellbinding region is capable of being selectively unmasked when thebispecific antibody is in the vicinity of the unwanted cells so as toallow binding to an immune cell. In this instance, the first portion ofthe bispecific antibody may correspond to the targeting moiety describedabove and the second portion of the bispecific antibody may correspondto the further moiety described above. Preferably, the antibody is asingle chain antibody construct; however, it is appreciated that theantibody may comprise two or more polypeptide chains, as discussedabove.

Preferably, the immune cell binding region of the bispecific antibody isa T cell binding region. Thus, the bispecific antibody may comprise (i)a first portion that is capable of specifically binding to an unwantedcell and (ii) a second portion that comprises a masked

T cell binding region so as to prevent binding of the second portion toa T cell, wherein the masked T cell binding region is capable of beingselectively unmasked when the bispecific antibody is in the vicinity ofthe unwanted cells so as to allow binding to a T cell. Typically, the Tcell binding region is one that can bind to the CD3 antigen and/or TCRpresent on a T cell.

It is also preferred that the immune cell binding region is unmasked byselective cleavage of one or more cleavage sites in the molecule when inthe vicinity of the unwanted cells. Thus, the bispecific antibody maycomprise (i) a first portion that is capable of specifically binding toan unwanted cell and (ii) a second portion that comprises a maskedimmune cell binding region so as to prevent binding of the secondportion to an immune cell, wherein the masked immune cell binding regionis unmasked by selective cleavage of one or more cleavage sites in themolecule when in the vicinity of the unwanted cells so as to allowbinding to an immune cell.

Accordingly, the invention provides a bispecific antibody comprising (i)a first portion that is capable of specifically binding to an unwantedcell and (ii) a second portion that comprises a masked T cell bindingregion so as to prevent binding of the second portion to a T cell,wherein the masked T cell binding region is unmasked by selectivecleavage of one or more cleavage sites in the molecule when in thevicinity of the unwanted cells so as to allow binding to a T cell.Typically, the T cell binding region is one that can bind to the CD3antigen and/or TCR present on a T cell.

Suitably, the targeting moiety, further moiety and, if present, the oneor more masking moieties are joined so that all moieties retain theirrespective activities such that the molecule may be targeted to anunwanted cell and the immune cell binding region may be unmasked withinthe vicinity of the unwanted cells so as to bind to an immune cell.

The targeting moiety and further moiety are typically joined by a linkerpeptide. Preferably, the linker joining the targeting moiety and furthermoiety contains between 2 and 100 amino acid residues, such as more thanor less than 10, 20, 30, 40, 50, 60, 70, 80 and 90 amino acids. Morepreferably, the linker joining the targeting moiety and further moietycontains between 2 and 50 amino acids and still more preferably between4 and 20 amino acids. Thus, the linker peptide may comprise 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids.

The one or more masking moieties are typically joined to the furthermoiety (including separate parts thereof) by a respective one or morelinker peptides. The lengths of such linker peptides may depend on howthe one or more masking moieties are operating to mask the T cellbinding region. For example, if the one or more masking moieties areimmunoglobulin domains that are intended to pair with variable domainsof a further moiety (e.g. FIG. 4), the linkers that join the one or moreimmunoglobulin domains of the masking moieties with the respectivevariable domains of the further moiety must be of a sufficient length toallow pairing between the respective immunoglobulin domains of the oneor more masking moieties and the respective variable domains of thefurther moiety. Generally, therefore, the linkers are at least 15 aminoacids in length, such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 aminoacids in length. If the one or more masking moieties are simply blockingaccess to the underlying T cell binding region of the further moiety(e.g. FIGS. 2 and 3), it will be appreciated that the one or morelinkers joining the respective one or more masking moieties to thefurther moiety may be shorter, or even zero length. Typical amino acidlengths include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 amino acids. However, longer linkers may also be used especially whenthe masking moiety is an immune cell surface antigen (e.g. FIG. 3).Thus, the linker may be at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acidsin length. If the one or more masking moieties are immunoglobulindomains that are intended to prevent pairing of variable domains of afurther moiety but without the immunoglobulin domains of the one or moremasking moieties pairing with the variable domains of the furthermoiety, the linkers that join the one or more immunoglobulin domains ofthe masking moieties with the respective variable domains of the furthermoiety must be of an insufficient length to allow pairing between therespective immunoglobulin domains of the one or more masking moietiesand the respective variable domains of the further moiety. Thus, thelinkers are generally 14 amino acids or less, such as 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 amino acid in length.

In some embodiments, the one or more masking moieties may be joined tothe targeting moiety (e.g. FIG. 4). Typically, the one or more maskingmoieties are joined to the targeting moiety by a respective one or morelinker peptides. Preferably, the linker joining the targeting moiety andfurther moiety contains between 2 and 100 amino acid residues, morepreferably between 2 and 50 and still more preferably between 4 and 20amino acids (eg at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 or 19 amino acids).

Suitable linker peptides are those that typically adopt a random coilconformation, for example the polypeptide may contain alanine or prolineor a mixture of alanine plus proline residues.

Polynucleotides which encode suitable targeting moieties and furthermoieties are known in the art or can be readily designed from knownsequences such as from sequences of proteins known to interact withsurface markers expressed on unwanted cells or immune cells or containedin nucleotide sequence databases such as the GenBank, EMBL,ImMunoGeneTics (IMGT), and dbEST databases. Polynucleotides which encodesuitable one or more masking moieties are also known in the art or canreadily be designed from known sequences or sequences in the abovedatabases and made.

Polynucleotides which encode suitable linker peptides can readily bedesigned from linker peptide sequences and made.

Thus, polynucleotides which encode the molecules used in the inventioncan readily be constructed using well known genetic engineeringtechniques.

The nucleic acid is then expressed in a suitable host to produce amolecule of the invention. Thus, the nucleic acid encoding the moleculeof the invention may be used in accordance with known techniques,appropriately modified in view of the teachings contained herein, toconstruct an expression vector, which is then used to transform anappropriate host cell for the expression and production of the moleculeof the invention of the invention.

It is appreciated that the nucleic acid encoding the molecule of theinvention may be joined to a wide variety of other nucleic acidsequences for introduction into an appropriate host. The companionnucleic acid will depend upon the nature of the host, the manner of theintroduction of the nucleic acid into the host, and whether episomalmaintenance or integration is desired, as is well known in the art.

In an alternative embodiment, the targeting moiety, further moiety(including separate parts thereof) and one or more masking moieties maybe non-covalently attached. For non-covalent bindings, immunologicalbindings or such binding as via biotin/avidin or streptavidin,respectively, are preferred. For example, the targeting moiety may be abispecific antibody, one specificity of which is directed to an entityexpressed by the unwanted cell and one specificity of which is directedto the further moiety or part thereof. Also, it is possible to couplethe further moiety to another substance against which, in turn, thespecificity of the bispecific antibody will be directed to. Forinstance, the further moiety may contain further peptidic sequenceswhich are recognised by the bispecific antibody. Another possibilityinvolves coupling the targeting moiety, for example to streptavidinwhilst the further moiety is coupled to biotin, and vice versa. Othermeans by which non-covalent interactions can be formed include leucinezipper sequences or affinity bonds. In any event, it is preferred thatthere are one or more cleavage sites within the molecule that arecleavable selectively when the molecule is in the vicinity of theunwanted cells, such that the immune cell binding region can beunmasked.

Although less preferred, the targeting moiety, further moiety (includingseparate parts thereof) and one or more masking moieties may beconveniently linked to each other by any of the conventional ways ofcross-linking molecules, such as those generally described in O'Sullivanet al Anal. Biochem. (1979) 100, 100-108. For example, one of thetargeting moiety or further moiety (including separate parts thereof) ormasking moiety or spacer moiety may be enriched with thiol groups andthe moiety it is to be linked to reacted with a bifunctional agentcapable of reacting with those thiol groups, for example theN-hydroxysuccinimide ester of iodoacetic acid (NHIA) orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aheterobifunctional cross-linking agent which incorporates a disulphidebridge between the conjugated species. Amide and thioether bonds, forexample achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, aregenerally more stable in vivo than disulphide bonds.

It is known that bis-maleimide reagents allow the attachment of a thiolgroup (e.g. thiol group of a cysteine residue of an antibody) to anotherthiol-containing moiety (e.g. thiol group of an immune cell antigen or alinker intermediate), in a sequential or concurrent fashion. Otherfunctional groups besides maleimide, which are reactive with a thiolgroup include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide,pyridyl disulfide, isocyanate, and isothiocyanate.

Further useful cross-linking agents include S-acetylthioglycolic acidN-hydroxysuccinimide ester (SATA) which is a thiolating reagent forprimary amines which allows deprotection of the sulphydryl group undermild conditions (Julian et al (1983) Anal. Biochem. 132, 68),dimethylsuberimidate dihydrochloride and N,N′-o-phenylenedimaleimide.

Particularly preferred crosslinking agents include sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC),sulfosuccinimidyl 6-(3′-[2-pyridyldithio]-propionamido) hexanoate(Sulfo-LC-SPDP) and N-[ß-Maleimidopropionic acid] hydrazide,trifluoroacetic acid salt (BMPH).

It will be understood that a large number of homobifunctional andheterobifunctional crosslinking chemistries would be appropriate to jointhe various moieties of the molecule together, and any such chemistrymay be used. For example, Click Chemistry using

Staudinger Ligation Chemistry (phosphine-azido chemistry) may be used.

Amino acid residues described herein are generally in the natural “L”isomeric form, which is preferred. However, residues in the “D” isomericform can be substituted for L-amino acid residues in certain situations,provided that the agent of the invention still retains its function,namely to prevent or treat a condition characterised by the presence ofunwanted cells. The definition also includes, unless otherwisespecifically indicated, chemically-modified amino acids, including aminoacid analogues (such as penicillamine, 3-mercapto-D-valine),naturally-occurring non-proteogenic amino acids (such as norleucine),beta-amino acids, azapeptides, N-methylated amino acids andchemically-synthesised compounds that have properties known in the artto be characteristic of an amino acid. The term “proteogenic” indicatesthat the amino acid can be incorporated into a protein in a cell throughwell-known metabolic pathways. The definition also includes amino acidsin which the functional side group has been chemically derivatised. Suchderivatised molecules include, for example, those molecules in whichfree amino groups have been derivatised to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatised to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatised to formO-acyl or O-alkyl derivatives. Also included as derivatives are thosepeptide portions that contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids.

Accordingly, it is appreciated that the peptide portions of the moleculeof the invention can be peptide “mimetics”, i.e. peptidomimetics whichmimic the structural features of peptides comprising or consisting ofthe amino acid sequence as described herein. Peptidomimetics can be evenmore advantageous in therapeutic use, in the resistance to degradation,in permeability or in possible oral administration.

A primary goal in the design of peptide mimetics has been to reduce thesusceptibility of mimetics to cleavage and inactivation by peptidases.In one approach, such as disclosed by Sherman et al (1990), one or moreamide bonds have been replaced in an essentially isosteric manner by avariety of chemical functional groups. This stepwise approach has metwith some success in that active analogues have been obtained. In someinstances, these analogues have been shown to possess longer biologicalhalf-lives than their naturally-occurring counterparts. In anotherapproach, a variety of uncoded or modified amino acids such as D-aminoacids and N-methyl amino acids have been used to modify mammalianpeptides. Alternatively, a presumed bioactive conformation has beenstabilised by a covalent modification, such as cyclization or byincorporation of γ-lactam or other types of bridges (Veber et al, 1978)and Thorsett et al, 1983). Another approach, disclosed by Rich (1986)has been to design peptide mimics through the application of thetransition state analogue concept in enzyme inhibitor design. Forexample, it is known that the secondary alcohol of statine mimics thetetrahedral transition state of the sessile amide bond of the pepsinsubstrate. Other approaches include the use of azapeptides andbeta-amino acids.

Also included in the definition of ‘peptidomimetics’, are retro-inversopeptides. By retro-inverso peptides (also known as all-D-retro orretro-enantio peptides) we include the meaning of a peptide in which allof the L-amino acids are replaced with D-amino acids and the peptidebonds are reversed. Thus, the peptides are composed of D-amino acidsassembled in the reverse order from that of the parent L-sequence.Retro-inverso peptides can be synthesised by methods known in the art,for example such as those described in Meziere et al (1997) J. Immunol.159 3230-3237. This approach involves making pseudopeptides containingchanges involving the backbone, and not the orientation of side chainswhich remain very similar to the parent peptide. Retro-inverse peptidesare much more resistant to proteolysis.

Therefore, it will be appreciated that when any of the targeting moiety,further moiety, one or more masking moieties, cleavage site, and spacermoieties as described herein are peptides or polypeptides, any one ormore of those peptides or polypeptides may be substituted for acorresponding peptidomimetic that retains the respective activity of theparent peptide or polypeptide. This may help to confer proteaseresistance on the agent of the invention and thereby improve itsstability. Thus, for example, when a targeting moiety is attached to afurther moiety via one or more peptide spacer moieties, it may bedesirable for one or more of those spacer moieties to bepeptidomimetics, e.g. wherein one or more of the naturally occurringamino acids of the spacer moieties are replaced or modified, forexample, to improve stability.

Another approach to increase stability of peptide portions of the agentof the invention is to have stabilising groups at one or both termini.Typical stabilising groups include amido, acetyl, benzyl, phenyl, tosyl,alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end groupmodifications. Additional modifications include using a “D” amino acidin place of a “L” amino acid at the termini, and amide rather than aminoor carboxy termini or acetyl rather than amino termini, to inhibitexopeptidase activity. Thus, it is appreciated that whenever the agentof the invention has an exposed peptide terminus, that terminus may havea capping moiety, preferably a moiety that is less than 200 Da inmolecular weight. Further capping moieties include a naftyl group or apolyethylene glycol group. It is appreciated that retro-inverso peptidesare already relatively stable and so may not require additional cappingmoieties.

Preferably, the molecule of the invention has a half-life in plasma ofat least 24 hours at 37° C.

It may be desirable to modify the molecule of the invention so that itcan be more easily detected, for example by biotinylating it or byincorporating any detectable label known in the art such as radiolabels,fluorescent labels or enzymatic labels.

As discussed above, the molecules of the invention have utility inredirecting the body's immune machinery to eradicate or neutraliseparticular unwanted cells in a specific manner. Since any unwanted cellmay be targeted in this way, the molecules of the invention offersignificant therapeutic potential.

Accordingly, a second aspect of the invention provides a method ofpreventing or treating a condition characterised by the presence ofunwanted cells, the method comprising administering a molecule accordingto the first aspect of the invention to a subject.

Thus, the method may involve identifying a subject who has a conditionor who is at risk of developing a condition characterised by unwantedcells (eg cancer), administering the molecule according to the firstaspect of the invention to the subject, and monitoring the levels of theunwanted cells in the subject either by conducting tests to determinethe number of unwanted cells or by monitoring the clinical symptoms ofthe subject. Depending on the results of the monitoring step, it may benecessary to administer more of the agent.

Similarly, the invention includes a molecule according to the firstaspect of the invention for use in preventing or treating a conditioncharacterised by the presence of unwanted cells.

The invention also includes the use of a molecule according to the firstaspect of the invention in the manufacture of a medicament forpreventing or treating a condition characterised by the presence ofunwanted cells.

Preferences for the condition and unwanted cells, are as described abovewith respect to the first aspect of the invention. Examples ofparticular conditions include tumours (benign or malignant), autoimmuneconditions, cardiovascular diseases, degenerative diseases, diabetes,allergic disease (eg asthma), neurodegenerative diseases such asAlzheimer's, transplantation patients and infectious diseases.Preferably, the unwanted cells are cancer cells and the condition is acancer. The cancer may be any cancer such as breast cancer, ovariancancer, endometrial cancer, cervical cancer, bladder cancer, renalcancer, melanoma, lung cancer, prostate cancer, testicular cancer,thyroid cancer, brain cancer, oesophageal cancer, gastric cancer,pancreatic cancer, colorectal cancer, liver cancer, leukaemia, myeloma,non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukaemia,acute lymphoblastic leukaemia, chronic lymphoblastic leukaemia,lymphoproliferative disorder, myelodysplastic disorder,myeloproliferative disease and premalignant disease. It will beappreciated that the molecule of the invention also has utility inregenerative medicine (eg laboratory grown organs or tissues).

By preventing or treating a condition we include the meaning of reducingor alleviating symptoms in a patient (i.e. palliative use), preventingsymptoms from worsening or progressing, treating the disorder (e.g. byinhibition or elimination of the causative agent), or prevention of thecondition or disorder in a subject who is free therefrom.

It will be appreciated that the molecules of the invention lendthemselves to personalised medicine in the clinic whereby the mostappropriate molecule to be administered to the patient is determined,and either selected or prepared in the clinic. For example, it may bedesirable to acquire an expression profile of the unwanted cell in apatient so that the optimum targeting moiety for that patient can beselected. Assessing the expression profile of the unwanted cell may becarried out on a biopsy sample using routine assays for measuringnucleic acid (e.g. DNA or RNA transcripts) or protein levels. Forexample, transcriptomic or proteomic techniques may be used. In thisway, it will be possible to identify tailored targeting moieties thatbind specifically to, for example, surface markers expressed by theunwanted cell. It may also be possible to identify appropriate proteasecleavage sites that may be selectively cleaved in the vicinity of theunwanted cells. For example, it may be that a molecule containing one ormore MMP2 cleavage sites would not be activated by a given patient'scancer proteases, but a molecule containing one or more MMP9 cleavagesites would be activated.

Thus the method of the second aspect of the invention may include thesteps of (i) identifying a subject who has a condition, or who is atrisk of developing a condition characterised by the presence of unwantedcells (eg cancer), (ii) taking a sample from the subject, (iii)analysing the sample to identify the optimum targeting moiety and/orcleavage site for preventing or treating the condition in that subject,(iii) preparing the molecule of the invention, (iv) administering themolecule to the subject, and (v) monitoring the levels of unwanted cellsin the subject either by conducting tests to determine the number ofunwanted cells or by monitoring the clinical symptoms of the subject.

It is appreciated that an apparatus may be used to select and optionallyprepare the most appropriate molecule to be used for a particularpatient. For example, the apparatus may perform an automated analysis ofone or more samples from the subject, and based on this analysis selectand optionally prepare a tailor-made molecule for that subject. Thus theapparatus may carry out an expression profile of unwanted cells from thesubject (eg from a biopsy sample) so as to determine a suitabletargeting moiety that will bind to the unwanted cell and/or determine asuitable cleavage site that will be selectively cleaved in the vicinityof the unwanted cell.

By performing any one or more of these steps in the clinic a moleculetailored for a particular subject can be prepared. For example, themolecule can contain a targeting moiety that is known to bindselectively to surface markers expressed by the unwanted cell, and/or acleavage site (e.g. protease cleavage site) that allows unmasking of theimmune cell binding region (eg T cell binding region) when the moleculeis in the vicinity of the unwanted cells.

In one embodiment, the subject is administered a further therapeuticagent in addition to the molecule according to the first aspect of theinvention. For example, when administering the molecule to prevent ortreat a particular condition, a further therapeutic agent known to beuseful for combating that condition may be administered. As an example,when the molecule is for treating cancer, a further anti-cancer agent(eg anti-neoplastic chemotherapy) may be administered to the subjectalongside the molecule of the invention. Similarly, the furthertherapeutic agent may be one that is known to have therapeuticapplication in allergic disease, inflammatory disease, regenerativemedicine and neuroregenerative disease.

It is appreciated that the further therapeutic agent may be administeredat the same time as the molecule of the invention (i.e. simultaneousadministration optionally in a co-formulation) or at a different time tothe molecule of the invention (i.e. sequential administration).

The further therapeutic agent may be any one or more of a vaccine; animmuno stimulatory drug; an anti-cancer agent; an agent inhibiting anantibody response against the agent of the invention; and/or a proteaseinhibitor.

For example, it may be desirable to administer immunostimulating agentssuch as IL-2, IL-7, IFNα, GM-CSF, metformin, lenalidomide; and/oradminister anti-immunoregulatory agents such as Ipilimumab; all of whichmay be considered as further therapeutic agents.

It is also appreciated that if the subject is one to whom isadministered immunosuppressive agents, that these immunosuppressiveagents may be withdrawn from the subject (e.g. by suspending treatment)when or before being administered the agent of the invention. This isparticularly true where the subject is one to whom is administeredimmunosuppressive agents to ablate T cells.

Similarly, it may be desirable to employ methods aimed at circumventingany immunogenicity issues relating to the molecule of the inventionwhereby an adverse antibody response is elicited in vivo. For example,the subject may also be administered one or more agents that are knownto inhibit the activity of B cells, such as any of Rituximab,cyclophosphamide, Syk inhibitors, an anti-BAFF antibody (eg Belimumab),an anti-CD22 antibody, an anti-CD20 antibody and an anti-CD19 antibody,all of which may be considered as further therapeutic agents. In thiscase, it is particularly preferred if the inhibitor of B cells isadministered to the subject prior to the molecule of the invention, egas a pre-treatment to ablate B cells.

In another embodiment, where the molecule comprises one or more proteasecleavage sites, it may be appropriate to administer a particularprotease inhibitor so as to improve the target selectivity of themolecule of the invention. For example, if a targeting moiety is knownto bind cells in both the heart and breast tissue, but only those in thebreast are to be targeted, it may be desirable to administer an agentthat selectively inhibits the protease responsible for unmasking theimmune cell binding region, in the heart but not the breast. In otherwords, an agent is administered to inhibit a protease that is capable ofunmasking the immune cell binding region but which protease resides inthe vicinity (eg at or near the surface of) of wanted cells but not inthe vicinity (eg at or near the surface of) of unwanted cells. This isparticularly useful in the event that a protease cleavage site withinthe molecule of the invention is cleavable by multiple proteases, someof which reside in the vicinity of unwanted cells and some of whichreside in the vicinity of wanted cells. In this case, targetingspecificity may be improved by administering a protease inhibitor thatinhibits a protease that resides in the vicinity of wanted cells butnevertheless is capable of cleaving the cleavage site and thereforeunmasking the immune cell binding region.

The effect of administering the inhibitor would be to ensure that theimmune cell binding region is preferentially unmasked in the vicinity ofthe unwanted cells. For instance, if a subject with cancer also hasactive rheumatoid arthritis where MMP2 and other proteases are active,and the one or more cleavage sites in the molecule of the invention arecleavable by multiple proteases including MMP2, it may be beneficial toinhibit MMP2 to prevent cleavage of the molecule at the arthritic jointbut retain cleavage at the cancer site by another protease.

The invention thus includes a composition comprising (i) a moleculeaccording to the first aspect of the invention and (ii) a furthertherapeutic agent, for use in preventing or treating a conditioncharacterised by the presence of unwanted cells. Given that the moleculeof the invention and the further therapeutic agent may be administeredsimultaneously or sequentially, it will be appreciated that theinvention includes a molecule according to the first aspect of theinvention for use in preventing or treating a condition characterised bythe presence of unwanted cells in a subject who is administered afurther therapeutic agent. It also follows that the invention includes atherapeutic agent for use in preventing or treating a conditioncharacterised by the presence of unwanted cells in a subject who isadministered a molecule according to the first aspect of the invention.

Similarly, the invention includes a use of a composition comprising (i)a molecule according to the first aspect of the invention and (ii) afurther therapeutic agent, in the manufacture of a medicament forpreventing or treating a condition characterised by the presence ofunwanted cells. Again, given that the molecule of the invention and thefurther therapeutic agent may be administered simultaneously orsequentially, it will be appreciated that the invention includes the useof a composition comprising a molecule according to the first aspect ofthe invention in the manufacture of a medicament for preventing ortreating a condition characterised by the presence of unwanted cells ina subject who is administered a further therapeutic agent. It alsofollows that the invention includes the use of a therapeutic agent inthe manufacture of a medicament for preventing or treating a conditioncharacterised by the presence of unwanted cells in a subject who isadministered a molecule according to the first aspect of the invention.

The invention also provides a composition comprising (i) a moleculeaccording to the first aspect of the invention and (ii) a furthertherapeutic agent suitable for preventing or treating the same conditioncharacterised by the presence of unwanted cells. It is appreciated thatthe therapeutic agent mentioned in the immediately preceding twoparagraphs may be agents suitable for treating the same conditioncharacterised by the presence of unwanted cells, as treatable by themolecules of the invention.

A third aspect of the invention provides a molecule according to thefirst aspect of the invention for use in medicine.

A fourth aspect of the invention a pharmaceutical composition comprisinga molecule according to the first aspect of the invention, and apharmaceutically acceptable carrier, diluent or excipient.

Whilst it is possible for the molecule of the invention to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe therapeutic agent and not deleterious to the recipients thereof.Typically, the carriers will be water or saline which will be sterileand pyrogen free.

Where appropriate, the formulations may conveniently be presented inunit dosage form and may be prepared by any of the methods well known inthe art of pharmacy. Such methods include the step of bringing intoassociation the active ingredient (agent for treating or preventing acondition characterised by unwanted cells) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The molecule of the invention can be administered in the form of asuppository or pessary, or they may be applied topically in the form ofa lotion, solution, cream, ointment or dusting powder. The molecule mayalso be transdermally administered, for example, by the use of a skinpatch.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The amount of the molecule which is administered to the individual is anamount effective to combat the particular individual's condition. Theamount may be determined by the physician.

Preferably, in the context of any aspect of the invention describedherein, the subject to be treated is a human. Alternatively, the subjectmay be an animal, for example a domesticated animal (for example a dogor cat), laboratory animal (for example laboratory rodent, for examplemouse, rat or rabbit) or an animal important in agriculture (i.e.livestock), for example horses, cattle, sheep or goats.

The invention provides a kit of parts for preventing or treating acondition characterised by the presence of unwanted cells, the kitcomprising:

-   -   (i) a targeting moiety capable of directly targeting to unwanted        cells and which is attached to a first binding partner, and    -   (ii) a further moiety that has a masked immune cell binding        region so as to prevent binding of the further moiety to an        immune cell, and which further moiety is attached to a second        binding partner which is capable of binding to the first binding        partner,    -   wherein the masked immune cell binding region is capable of        being selectively unmasked when the further moiety is in the        vicinity of the unwanted cells so as to allow binding of the        further moiety to an immune cell. Preferably, the immune cell        binding region is a T cell binding region (eg one that binds to        CD3 antigen and/or TCR on a T cell). Preferably, the immune cell        binding region is unmasked by selective cleavage of one or more        cleavage sites in the further moiety when in the vicinity of the        unwanted cells.

Preferences for the unwanted cells, targeting moiety, further moiety andcleavage sites are as defined above. It is particularly preferred if thekit of parts is for preventing or treating cancer. It is alsoappreciated that the kit of parts may comprise one or more maskingmoieties that mask the immune cell binding region of the further moiety.Preferences for the masking moiety are as defined above, and includeboth masking moieties that promote a conformation of the further moietyin which conformation the immune cell binding region is not accessibleto an immune cell, as well as masking moieties that simply block anunderlying immune cell binding region of the further moiety.

By the first and second binding partners, we include the meaning of anytwo moieties which bind to each other selectively. Most preferably, thefirst and second binding partners only bind to each other and not to anyother moieties. Non-covalent binding such as between biotin/avidin orstreptavidin, or immunological bindings are preferred. Thus, the firstbinding partner may be biotin and the second binding partner may beavidin, and vice versa. Alternatively, the first binding partner may bean antigen and the second binding partner may be an antibody specificfor that antigen, and vice versa. However, any pair of first and secondbinding partners that selectively bind to each other may be used, andsuitable pairs will be known to the skilled person.

It will be appreciated that the kit allows one to first administer thetargeting moiety that directly targets the unwanted cells to thesubject, and establish the correct localisation of the targeting moietyin the subject (for example by the targeting moiety being detectablylabelled (eg radiolabelled)) before administering the further moiety.The further moiety is then targeted to the unwanted cells by virtue ofthe second binding partner binding to the first binding partner. Once inthe vicinity of the unwanted cells, the immune cell binding region ofthe further moiety is unmasked, for example by selective cleavage of oneor more cleavage sites within the further moiety, allowing an immunecell (eg T cell) to be recruited to the unwanted cells. It will beunderstood that the further moiety that is attached to a second bindingpartner may be considered a targeting moiety that is capable ofindirectly targeting to an unwanted cell as mentioned above in relationto the first aspect of the invention.

Accordingly, the invention further provides a method of preventing ortreating a condition characterised by the presence of unwanted cells,the method comprising administering

-   -   (i) a targeting moiety capable of directly targeting to unwanted        cells and which is attached to a first binding partner, and    -   (ii) a further moiety that has a masked immune cell binding        region so as to prevent binding of the further moiety to an        immune cell, and which further moiety is attached to a second        binding partner which is capable of binding to the first binding        partner,    -   wherein the masked immune cell binding region is capable of        being selectively unmasked when the further moiety is in the        vicinity of the unwanted cells so as to allow binding of the        further moiety to an immune cell. Preferably, the immune cell        binding region is a T cell binding region (eg one that binds to        CD3 antigen and/or TCR on a T cell). It is also preferred that        the immune cell binding region is unmasked by selective cleavage        of one or more cleavage sites in the further moiety when in the        vicinity of the unwanted cells. Preferably, the targeting moiety        is administered before the further moiety, for example to allow        to correct localisation of the targeting moiety at the unwanted        cells to be established. However, the targeting moiety may be        administered at the same time as the further moiety. It is        appreciated that the targeting moiety and further moiety may be        attached to each other by binding of the first and second        binding partners.

Similarly, the invention provides a composition comprising:

-   -   (i) a targeting moiety capable of directly targeting to unwanted        cells and which is attached to a first binding partner, and    -   (ii) a further moiety that has a masked immune cell binding        region so as to prevent binding of the further moiety to an        immune cell, and which further moiety is attached to a second        binding partner which is capable of binding to the first binding        partner,    -   wherein the masked immune cell binding region is capable of        being selectively unmasked when the further moiety is in the        vicinity of the unwanted cells so as to allow binding of the        further moiety to an immune cell,    -   for use in preventing or treating a condition characterised by        unwanted cells in a subject. Preferably, the immune cell binding        region is a T cell binding region (eg one that binds to CD3        antigen and/or TCR on a T cell). It is also preferred that the        immune cell binding region is unmasked by selective cleavage of        one or more cleavage sites in the further moiety when in the        vicinity of the unwanted cells

Similarly, the invention provides a use of a composition comprising:

-   -   (i) a targeting moiety capable of directly targeting to unwanted        cells and which is attached to a first binding partner, and    -   (ii) a further moiety that has a masked immune cell binding        region so as to prevent binding of the further moiety to an        immune cell, and which further moiety is attached to a second        binding partner which is capable of binding to the first binding        partner, wherein the masked immune cell binding region is        capable of being selectively unmasked when the further moiety is        in the vicinity of the unwanted cells so as to allow binding of        the further moiety to an immune cell,    -   in the manufacture of a medicament for preventing or treating a        condition characterised by the presence of unwanted cells.        Preferably, the immune cell binding region is a T cell binding        region (eg one that binds to CD3 antigen and/or TCR on a T        cell). It is also preferred that the immune cell binding region        is unmasked by selective cleavage of one or more cleavage sites        in the further moiety when in the vicinity of the unwanted        cells.

In an embodiment of the invention, the targeting moiety is an anti-MUC1antibody, and the further moiety is an anti-CD3 antibody.

In an embodiment of the invention, the targeting moiety is an anti-CD19antibody, and the further moiety is an anti-CD3 antibody.

The invention will be described in further detail with the aid of thefollowing Figures and Examples.

FIG. 1: Schematic diagram illustrating molecule of the invention. Thetargeting moiety (1) comprises VH and VL domains specific for anunwanted cell (2), and the further moiety (3) comprises VH and VLdomains specific for CD3 antigen. The VH and VL domains of the furthermoiety are attached by a linker which is of insufficient length to allowpairing, and so the T cell binding region of the further moiety ismasked. Cleavage of a cleavage site (5) in the linker when the moleculeis in the vicinity of the unwanted cell (2) allows unmasking of the Tcell binding region (4).

FIG. 2: Schematic diagram illustrating molecule of the invention. Thetargeting moiety (1) comprises paired VH and VL domains specific for anantigen on the unwanted cell (2). The further moiety (3) comprises VHand VL domains specific for CD3 antigen. However, the T cell bindingregion of the further moiety is blocked by the presence of two maskingmoieties, a CH domain and a CL domain (6) that are joined to the furthermoiety by respective linkers. Cleavage of cleavage sites (5) in thelinkers when the molecule is in the vicinity of the unwanted cell (2)allows unmasking of the T cell binding region (4).

FIGS. 3A-3B: Schematic diagram illustrating molecule of the invention.The targeting moiety (1) comprises paired VH and VL domains specific foran antigen on the unwanted cell (2). The further moiety (3) comprises VHand VL domains specific for CD3 antigen. However, the T cell bindingregion of the further moiety is blocked by the presence of a maskingmoiety (6) which is an immune cell antigen that binds to the T cellbinding region, and which is securely tethered to the further moiety bya covalent linker. Cleavage of a cleavage site (5) in the linker whenthe molecule is in the vicinity of the unwanted cell (2) allows themasking moiety to leave the T cell binding region and so the T cellbinding region (4) is unmasked.

FIG. 4: Schematic diagram illustrating molecule of the invention. Thetargeting moiety (1) comprises paired VH and VL domains specific for anantigen on the unwanted cell (2). The further moiety (3) comprises VHand VL domains specific for CD3 antigen. However, the VH and VL domainsof the further moiety (3) are unable to pair due to the presence of twomasking moieties (6) respectively comprising a VL and a VH domain. TheVL and VH domains of the masking moieties act as dummy domains that pairwith the VH and VL domains of the further moiety and so prevent the VHand VL domains of the further moiety from pairing. The T cell bindingregion (4) is therefore masked. Cleavage of cleavage sites (5) inlinkers that join the further moiety to the one or more masking moietieswhen the molecule is in the vicinity of the unwanted cell (2) releasesthe VH and VL domains from pairing with the dummy VH and VL domains ofthe masking moiety and so the VH domain and VL domain of the furthermoiety can pair. The T cell binding region (4) is thereby unmasked.

FIGS. 5A-5B: (A) Schematic diagram illustrating a molecule of theinvention. The VH and VL regions encompassing the scFv of the targetingmoiety are linked via a single peptide linker to the VH region of thefurther moiety which in turn is linked via a single peptide linkercontaining a protease cleavage site to the CH region of the maskingmoiety. The VL region of the further moiety is linked via a singlepeptide linker containing a protease cleavage site to the CL region ofthe masking moiety. The VH and VL regions of the further moiety and theCH and CL regions of the masking moiety will pair when the proteins areincubated together. Thus, the CH and CL regions of the masking moietyprevent the VH and VL regions of the further moiety from interactingwith its natural ligand (eg CD3) and from activating T cells. However,after binding to unwanted cells via the target moiety, specificproteolytic cleavage will occur in the vicinity of unwanted cells andthe masking moiety will be thereby released allowing interaction of Tcells with the unwanted cell. (B) Schematic diagram denoting the geneticmake-up of the protein chains depicted in (A). Rickymab (Heavy)comprises the VH of the further moiety and the CH region of the maskingmoiety and this will be molecularly cloned together with the scFv of thetarget moiety. Rickymab (Light) comprises the VL region of the furthermoiety and the CL region of the masking moiety which will pair with theVH and CH regions of Rickymab (Heavy) after introducing the DNA intocells capable of generating the embodiment.

FIGS. 6A-6B: (A) Schematic diagram that denotes the regions of DNAcombined together to form the polypeptide chain seen in FIG. 4. Furtherindications by arrow denote protease cleavage sites and sites of DNAenzyme cleavage (BamH, Sac & Xho) allowing for movement of regionswithin the molecule to generate the molecules seen in FIGS. 1, 2, 4 and5A-5B. (B) Schematic diagram that denotes the regions of DNA that makeup the VH and CH regions of the target moiety in (A) coupled to afluorescent dye. The VH and CH regions of the other molecules (eg FIGS.1-5B) could also be linked to the fluorescent dye by enzymatic digestionof the DNA and subsequent ligation to the fluorescent dye. Once thelabelled molecule is used to label cells, those cells can be viewedusing flow cytometry. This allows demonstration that the target moietybinds to the unwanted cells of interest and not any wanted cell types.

FIGS. 7A-7F: Peripheral blood mononuclear cells (PBMCs) from 2 donors(with different HLA types) were incubated with the molecule exemplifiedin FIG. 3 (comprising an anti-CD19 scFv (targeting moiety) linked to ananti-CD3 scFv (the further moiety) further linked to the CD3 epsilondomain (the masking moiety)—B.mab) and incubated overnight at 37° C.with the addition of a protease to release the active site of thefurther moiety. The following day, cells were labelled with antibodiesagainst anti-CD3 to determine the presence of T cells after incubationwith the molecule and also anti-CD19 to determine the presence/absenceof B cells (see Example 2). Incubation with the molecule removes all Bcells from the PBMCs as would be expected.

FIG. 8: Target cells, B lymphoblastoid cell line (B-LCL)) are labelledwith a molecule comprising an anti-CD19 scFv (targeting moiety) linkedto an anti-CD3 scFv (the further moiety) further linked to the CD3epsilon domain (the masking moiety) (FIG. 3). After removing unboundembodiment, the B-LCL cells were incubated overnight with or without Tcells with the B-LCLs producing the protease which cleaves the maskingmoiety releasing the further moiety. As a control measure, the T cellswere incubated with the molecule without target cells to determinebackground activation of unbound molecule. After overnight incubation,the amount of interferon gamma (IFN-γ) in each culture was assayed todetermine the extent of T cell activation. There was only activation ofT cells when target cells were labelled with the molecule (see Example1).

FIGS. 9A-9F: Conditionally activated bispecific construct. Peripheralblood mononuclear cells from a healthy donor were assessed using flowcytometry following overnight culture. Cells were stained using anti-CD3(Y-axis) and anti-CD19 (x-axis) to reveal numbers of T-cells andB-cells. The scFv1-scFv2-CD3e construct contained a protease cleavablesection between the CD3e cap and the scFv1-scFv2 activating region whichcould be cleaved using the Factor Xa protease. scFv1 is the anti-CD19entity while scFV2 has affinity toward CD3e. (A) PBMCs culturedovernight in media only; (B) PBMCs+scFv1-scFv2-CD3e construct; (C)PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.01 ug/ml); (D)PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.1 ug/ml); (E)PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.5 ug/ml); (F)PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (1 ug/ml). As can beseen from the figure, only when the protease is added is the cap cleavedoff, thereby allowing depletion of B cells. The construct comprised ananti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (thefurther moiety) further linked to the CD3 epsilon domain (the maskingmoiety).

FIGS. 10A-10E: Conditionally activated bispecific construct throughremoval of masking domain. (A) Design of 2 constructs Ricky2A (SEQ IDNo: 30) and Ricky2B (SEQ ID No: 31) which when co-expressed in mammalianexpression systems (293T) produce a scFv1-scFv2-Cap where the anti-CD3binding region (scFv2) is masked by an immunoglobulin constant region(CH,CL). The constant region is cleaved by tumour-associated proteases(MMP2) through inclusion of an MMP2 recognition site IPVSLR (SEQ ID No:32) between the masking and binding domains. In this case an anti-MUC1(scFv1) and anti-CD3 (scFv2) domains are used for the construct; (B)T-cells used for experiment stained using CD3-PE (x-axis) andanti-CD138-APC (y-axis) as assessed using flow cytometry; (C)Muc1-expressing U266 cells used for experiment stained using CD3-PE(x-axis) and anti-CD138-APC (y-axis) as assessed using flow cytometry;(D) Co-culture of U266 cells with T-cells; (E) Co-culture of U266 cellswith T-cells+Ricky2A/2B activatable masked bispecific showing 12%reduction of U266 population.

EXAMPLE 1: USE OF MOLECULE OF INVENTION

Target cells modelling B cell malignancy (B lymphoblastoid cell line(B-LCL)) are targeted with a molecule comprising an anti-CD19 scFv(targeting moiety) linked to an anti-CD3 scFv (the further moiety)further linked to the CD3 epsilon domain (the masking moiety) (egmolecule in FIG. 3). Once targeted, excess molecule is washed from thetarget cells and the target cells are cultured with T cells (CD4+ andCD8+) overnight at 37° C. After incubation, supernatant from the cultureis tested for IFN-gamma release which would demonstrate the activationof the T cells in recognising the molecule on the surface of the targetB-LCLs. Activation of T cells would only be seen if proteolytic cleavageof the linker between the further moiety and CD3 epsilon masking moietyhas occurred, thus unmasking the binding region of the anti-CD3 bindingregion. After incubation of the molecule with either target cells aloneor with T cells alone, supernatant from the cultures demonstrated noIFN-gamma production. However, after incubation of targeted B-LCLs withT cells, there was a large production of IFN-gamma demonstrating therelease of the masking moiety in the presence of unwanted target cells(B-LCL) and the recognition of the unwanted cells by T cells.

EXAMPLE 2: USE OF MOLECULE OF INVENTION

Targeting B cells in a mixed population of human peripheral bloodmononuclear cells (containing B cells, T cells, monocytes, macrophagesand NK cells), with a molecule comprising an anti-CD19 scFv (targetingmoiety) linked to an anti-CD3 scFv (the further moiety) further linkedto the CD3 epsilon domain (the masking moiety). Once the molecule hasbeen added to the mixed population of cells, thereby labelling B cells,an exogenous protease (e.g. trypsin) is added to the cells to releasethe masking agent before culturing at 37° C. overnight. After culture,the cells are labelled with fluorescently labelled antibodies anti-CD3and anti-CD19 and analysed using flow cytometry. The results demonstratethe presence of B cells (˜10%) in the cells after overnight culturewithout the molecule but the complete ablation of B cells in the cellscultured overnight in the presence of the molecule. This resultdemonstrates the targeted cytotoxicity of unwanted B cells in a mixedpopulation of peripheral blood mononuclear cells.

EXAMPLE 3: SYNTHESIS OF MOLECULE OF INVENTION

The production of the molecules shown in the figure comprises generatingan protein sequence of the target moiety (VH and VL domains) and theprotein sequence of the further moiety (VH and VL domains) and linkingthese domains using standard proteomic linkages (Gly-Gly-Gly-Serrepeats: SEQ ID No: 33). The scFv domains may then be linked to one ormore masking moieties using similar techniques. Where required, one ormore protease cleavage sequences can be inserted into the linkerpolypeptide sequences. Once the protein sequence has been determined forone or more polypeptide chains, the DNA is synthesised and inserted intoa vector suitable for either prokaryotic or eukaryotic expressionsystems. In a prokaryotic system, E. coli are transformed with thesynthesised gene encoding one or more polypeptide chains and afterculture overnight, the supernatant is harvested and the polypeptidechains can be purified by standard techniques (e.g. His tagpurification). After purification, the one or more polypeptide chainsare refolded and can then be used to label target cells. In a eukaryoticexpression system, the synthesised gene encoding one or more polypeptidechains is transfected into a suitable cell line in a lentiviralexpression system. After 48 hours incubation, supernatant containing thecorrectly refolded and conformationally correct molecules can bepurified using standard techniques (e.g. His tag purification).

An example of a molecule depicted in FIG. 3 contains the following aminoacid sequences on a single polypeptide chain:

Anti-CD19 VL (SEQ ID No: 20)DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPW TFGGGTKLEIK Linker(SEQ ID No: 21) GGGGSGGGGSGGGGS Anti-CD19 VH (SEQ ID No: 22)QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS Linker (SEQ ID No: 23) GGGGSSD Anti-CD3 VH(SEQ ID No: 24) IKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHMKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSLinker (SEQ ID No: 25) VEGGSGGSGGSGGSGGVDD Anti-CD3 VL (SEQ ID No: 26)IQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGT KLELK Long Linker(SEQ ID No: 21) GGGGSGGGGSGGGGS Cleavage site (SEQ ID No: 27) TIPVSLRLong Linker (SEQ ID No: 28) SGGGGSGGGGSGGGGSDI CD3 epsilon(SEQ ID No: 29) QTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCEGS

This molecule is illustrative of an embodiment of the invention wherethe molecule comprises one or more masking moieties that are immune cellsurface antigens, in this case CD3 epsilon.

An example of a molecule depicted in FIG. 5 is shown in FIG. 10. Thismolecule is illustrative of an embodiment of the invention where themolecule comprises one or more masking moieties which are immunoglobulindomains, and more specifically, constant domains. The embodiment is alsoillustrated in FIG. 2, but whereas in FIG. 2, the VH and VL portions ofthe targeting moiety are on separate polypeptide chains, in FIG. 5, theVH and VL portions of the targeting moiety are on the same polypeptidechain.

What is claimed is:
 1. A method of preventing or treating a tumour, themethod comprising (i) administering to a subject a molecule forredirecting T cells to tumour cells or (ii) administering to a subject amolecule for redirecting T cells to tumour cells and a furthertherapeutic agent, which may include one or more of an immunostimulatorydrug, an anti-cancer agent, and an inhibitor of an antibody responseagainst the molecule of the invention, wherein the molecule forredirecting T cells to tumor cells comprises: (a) a targeting moietycapable of targeting to tumour cells wherein the targeting moiety is anantibody or an antigen-binding fragment thereof that specifically bindsto an antigen expressed by the tumour cells and wherein the targetingmoiety is not masked, and (b) at least one further moiety comprisingcomponents of a T-cell binding region joined by at least one linker tothe targeting moiety, wherein the T-cell binding region is masked so asto prevent binding of the further moiety to a T cell, wherein the maskedT-cell binding region is capable of being selectively unmasked bycleavage of at least one protease cleavage site when the molecule is inthe vicinity of the tumour cells so as to allow binding of the furthermoiety to a T cell and wherein the further moiety comprises separately afirst VH and a first VL domain which when paired are capable ofspecifically binding to a T cell, a linker comprising at least oneprotease cleavage site which joins the first VH domain to a second VLdomain of a first masking moiety, and a linker comprising at least oneprotease cleavage site which joins the first VL domain to a second VHdomain of a second masking moiety, and wherein the linker which joinsthe first VH domain to the second VL domain is of a sufficient length toallow pairing of the first VH domain to the second VL domain, andwherein the linker which joins the first VL domain to the second VHdomain is of a sufficient length to allow pairing of the first VL domainto the second VH domain, such that the first VH and first VL domains ofthe further moiety are not paired and the further moiety cannot bind tothe T cell, and wherein selective cleavage of the protease cleavagesites, when in the vicinity of the tumour cells, allows pairing of thefirst VH and first VL domains such that the further moiety can bind tothe T cell.
 2. A method according to claim 1, wherein the tumour ischosen from leukaemia, lymphoma, sarcoma, or carcinoma.
 3. A methodaccording to claim 1, wherein the targeting moiety antibody orantigen-binding fragment thereof is specific for any of Her2/Neu; CD22;EpCAM/CD326; EGFR; PMSA; CD30; CD20; CD33; membrane IgE; IgEReceptor/CD23; CD80; CD86; CD2; CA125; Carbonic Anhydrase IX; CD70;CD74; CD56; CD40; CD19; c-met/HGFR; TRAIL-R1; DR5; PD-1; PDL1; IGF-1R;VEGF-R2; Prostate stem cell antigen (PSCA); MUC1; CanAg; Mesothelin;P-cadherin; Myostatin/GDF8; Cripto/TDGF1; ACVRL1/ALK1; MUCSAC; CEACAM;SLC44A4; CS1; CD137; CXCR4; Neuropilin 1; Glypican; HER3; PDGFRa andEphA2.
 4. A method according to claim 1, wherein the targeting moietyantibody or antigen-binding fragment thereof is an anti-epidermal growthfactor receptor antibody, an anti-Her2 antibody, an anti-CD20 antibody,an anti-CD22 antibody, an anti-CD70 antibody, an anti-CD33 antibody, ananti-MUC1 antibody, an anti-CD40 antibody, an anti-CD74 antibody, ananti-P-cadherin antibody, an anti-EpCAM antibody, an anti-CD138antibody, an anti-E-cadherin antibody, an anti-CEA antibody, and ananti-FGFR3 antibody.
 5. A method according to claim 1, wherein thetargeting moiety antibody or antigen binding fragment thereof is a scFvantibody, a camelid antibody, or an engineered camelid antibody.
 6. Amethod according to claim 1, wherein each of the further moiety andtargeting moiety are parts of a single polypeptide chain.
 7. A methodaccording to claim 1, wherein the targeting moiety antibody orantigen-binding fragment thereof is Cetuximab, Rituximab, Inotuzumab, orGemtuzumab.
 8. A method according to claim 1, wherein the targetingmoiety antibody or antigen-binding fragment thereof is a full-lengthantibody.
 9. A method according to claim 1, wherein the targeting moietyantibody or antigen-binding fragment thereof is an antigen-bindingfragment of an antibody.
 10. A method according to claim 1, wherein thetargeting moiety antibody or antigen-binding fragment thereof is a humanor humanized antibody or antigen binding fragment thereof.
 11. A methodaccording to claim 1, wherein the targeting moiety antibody orantigen-binding fragment thereof is a human scFv or a humanized scFv.12. A method according to claim 1, wherein the first VH and first VLdomains of the further moiety comprise an antibody specific for CD3. 13.A method according to claim 1, wherein the first VH and first VL domainsof the further moiety comprise an antibody specific for the TCR.
 14. Amethod according to claim 1, wherein the linker joining the first VHmoiety to the second VL moiety and the linker joining the first VLmoiety to the second VH moiety are from 15 to 40 amino acids in length.